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Antibody-mediated rejection of the liver allograft: An update and a clinico-pathological perspective

  • Brian T. Lee
    Correspondence
    Corresponding authors. Address: One Gustave Levy Place-Box 1104, New York, NY 10029, USA; Tel.: (212) 659-8502, fax (212) 241-2138.
    Affiliations
    Division of Gastroenterology and Transplant Institute, Loma Linda University Health, Loma Linda, CA, USA
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  • M. Isabel Fiel
    Affiliations
    Department of Pathology, Molecular and Cell-Based Medicine, Recanati/Miller Transplantation Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Thomas D. Schiano
    Correspondence
    Corresponding authors. Address: One Gustave Levy Place-Box 1104, New York, NY 10029, USA; Tel.: (212) 659-8502, fax (212) 241-2138.
    Affiliations
    Division of Liver Diseases, Department of Medicine, Recanati/Miller Transplantation Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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Published:August 02, 2021DOI:https://doi.org/10.1016/j.jhep.2021.07.027

      Summary

      Antibody-mediated rejection after liver transplantation is an under-recognised cause of allograft injury. While definitions of acute and chronic antibody-mediated rejection have increased clinical awareness, timely identification and management of antibody-mediated rejection remain difficult because of complexities in diagnosis and histopathology, lack of treatment protocols, and unclear long-term outcomes. While recent cohort studies assessing the importance of donor-specific antibodies have aided in its diagnosis, literature on the treatment of antibody-mediated rejection in liver transplantation remain limited to case reports and small series. Further increasing the awareness and timely recognition of antibody-mediated rejection post-liver transplantation is crucial in order to stimulate future research and the development of protocols for its diagnosis and treatment. This review will summarise recent advances in the clinical diagnosis and treatment of antibody-mediated rejection in liver transplantation, as well as some of the histopathologic features (on liver biopsy tissue) of acute and chronic antibody-mediated rejection.

      Keywords

      Introduction

      As direct-acting antivirals have eliminated recurrent HCV infection after liver transplantation (LT),
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      • Li A.A.
      • March K.L.
      • Yoo E.R.
      • Kim D.
      • Snyder H.
      • et al.
      Improved outcomes in HCV patients following liver transplantation during the era of direct-acting antiviral agents.
      transplant clinicians are more focused on maintaining durable allograft health. Barriers to successful long-term LT outcomes such as antibody-mediated rejection (AMR) remain incompletely understood. Traditionally, AMR was thought to be an infrequent complication post-LT with minimal impact on graft survival,
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      but its true incidence remains unknown. In 2013, a survey of 41 US centres assessed general perspectives regarding AMR in LT.
      • Neil D.A.H.
      • Bellamy C.O.
      • Smith M.
      • Haga H.
      • Zen Y.
      • Sebagh M.
      • et al.
      Global quality assessment of liver allograft C4d staining during acute antibody-mediated rejection in formalin-fixed, paraffin-embedded tissue.
      While 91% of respondents believed that AMR exists, only 46% believed it was a cause of allograft injury. Since then, there has been a resurgence of interest in AMR after LT, including diagnostic strategies involving donor-specific antibodies (DSAs), standardising liver histology, and varied approaches to management. Hence, in 2016, The Banff Working Group included AMR in its update on liver transplant pathology.
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      The following review summarises the recent diagnostic and histopathologic data on AMR post-LT.

      The liver as an “immune-privileged” organ

      While the true incidence of AMR in LT is unknown due to under-recognition, it is estimated to be lower (1% of LT recipients)
      • Demetris A.J.
      • Zeevi A.
      • O'Leary J.G.
      ABO-compatible liver allograft antibody-mediated rejection: an update.
      than in heart (10–20%)
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      • Ross H.J.
      • Tinckam K.
      • Butany J.
      • Rao V.
      Antibody-mediated rejection: an evolving entity in heart transplantation.
      and kidney (20–50%) transplantation (KT)
      • Gloor J.
      • Stegall M.D.
      Sensitized renal transplant recipients: current protocols and future directions.
      due to the unique anatomy of the liver and its identity as an “immune-privileged” organ.
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      The dual blood supply of the liver leads to a low flow state within the fenestrated sinusoidal system, allowing for the facilitation of interactions among intestinal microbial products, antigens, and immune cells while accommodating this ongoing exposure without sensitisation.
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      The sheer volume of the liver parenchyma allows for an increased endothelial surface for interactions with antibodies.
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      Human leukocyte antigen (HLA) class I antigens are expressed on all cells in the liver, while HLA class II expression on antigen-presenting cells is relatively low compared to other organs.
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      ,
      • O'Leary J.G.
      • Demetris A.J.
      • Friedman L.S.
      • Gebel H.M.
      • Halloran P.F.
      • Kirk A.D.
      • et al.
      The role of donor-specific HLA alloantibodies in liver transplantation.
      In addition, the liver sinusoids are lined by Kupffer cells and endothelial cells. These cells enable the capture and clearance of circulating immune complexes of HLA class I molecules and alloantibodies, allowing for regulation of immune responses and potentially building resistance to AMR.
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      The effects of this immune privilege can be seen in lower rejection rates with simultaneous kidney, intestine, and heart allografts.
      • Fong T.L.
      • Bunnapradist S.
      • Jordan S.C.
      • Selby R.R.
      • Cho Y.W.
      Analysis of the United Network for Organ Sharing database comparing renal allografts and patient survival in combined liver-kidney transplantation with the contralateral allografts in kidney alone or kidney-pancreas transplantation.
      • Abu-Elmagd K.M.
      • Costa G.
      • Bond G.J.
      • Soltys K.
      • Sindhi R.
      • Wu T.
      • et al.
      Five hundred intestinal and multivisceral transplantations at a single center: major advances with new challenges.
      • Wong T.W.
      • Gandhi M.J.
      • Daly R.C.
      • Kushwaha S.S.
      • Pereira N.L.
      • Rosen C.B.
      • et al.
      Liver allograft provides immunoprotection for the cardiac allograft in combined heart-liver transplantation.
      Finally, the regenerative capacity and ability to ameliorate fibrogenesis are unique to the liver in organ transplantation.
      • Demetris A.J.
      • Bellamy C.O.
      • Gandhi C.R.
      • Prost S.
      • Nakanuma Y.
      • Stolz D.B.
      Functional immune anatomy of the liver-as an allograft.
      This immune privilege differs from the concept of “immune-tolerance” in LT recipients, which can be achieved by steady weaning of immunosuppression (known as “operational tolerance”) in the setting of liver tolerogenesis through microchimerism
      • Starzl T.E.
      • Demetris A.J.
      • Trucco M.
      • Murase N.
      • Ricordi C.
      • Ildstad S.
      • et al.
      Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance.
      and lymphocyte senescence.
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      • Gibbs P.
      • Akbar A.N.
      • et al.
      Features of immune senescence in liver transplant recipients with established grafts.
      Despite the liver’s immune privilege, recipients remain susceptible to immune-mediated injury whether this is related to insufficient immunosuppression, recurrent disease (e.g. autoimmune, viral hepatitis), subclinical acute rejection, or extrahepatic diseases resulting in modulation of immunosuppression (e.g. solid organ malignancy, post-transplant lymphoproliferative disease). This is of particular concern as we strive to achieve the balance between safely withdrawing immunosuppression and optimising durable allograft function.

      Definitions of acute and chronic antibody-mediated rejection in liver transplantation

      Acute antibody-mediated rejection

      The mechanism for AMR of the liver allograft has been described by a “two-hit” hypothesis.
      • Kim P.T.
      • Demetris A.J.
      • O'Leary J.G.
      Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.
      An initial insult to the allograft (e.g. T cell-mediated rejection [TCMR], viral hepatitis, hepatic ischaemia, ischaemia-reperfusion injury) upregulates HLA class II expression, thus facilitating DSA binding and activating the classical complement cascade, while also facilitating cellular cytotoxicity through Fc receptor interactions, resulting in graft injury.
      • Valenzuela N.M.
      • Reed E.F.
      Antibody-mediated rejection across solid organ transplants: manifestations, mechanisms, and therapies.
      While the liver is considered an “immune-privileged” organ, AMR of the liver allograft is possible and prevalent, although underestimated by clinicians.
      While acute AMR has been widely described as mild allograft dysfunction, anecdotal presentations of hyper-acute allograft failure (e.g., haemorrhagic liver necrosis within weeks after LT) have been reported; the current challenge in liver AMR involves subclinical presentations of allograft dysfunction due to either decreased awareness by transplant providers or mitigation by more potent immunosuppressive regimens than those adopted in the past.
      The definition of acute AMR encompasses the following 4 criteria
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      : i) Histology: endothelial cell hypertrophy (Fig. 1A), portal capillary dilatation, microvasculitis with monocytes, eosinophils and neutrophils (Fig. 1B), and portal/peri-portal oedema – microvascular involvement of the central veins can distinguish acute AMR from other types of injury early after LT
      • O'Leary J.G.
      • Michelle Shiller S.
      • Bellamy C.
      • Nalesnik M.A.
      • Kaneku H.
      • Jennings L.W.
      • et al.
      Acute liver allograft antibody-mediated rejection: an inter-institutional study of significant histopathological features.
      ; ii) Elevated DSAs; iii) Diffuse C4d deposition of microvasculature in ABO-compatible tissues, or portal stroma in ABO-incompatible tissues; iv) Exclusion of other liver diseases or complications that can cause similar patterns of injury (i.e., concomitant TCMR).
      • O'Leary J.G.
      • Michelle Shiller S.
      • Bellamy C.
      • Nalesnik M.A.
      • Kaneku H.
      • Jennings L.W.
      • et al.
      Acute liver allograft antibody-mediated rejection: an inter-institutional study of significant histopathological features.
      • O'Leary J.G.
      • Kaneku H.
      • Demetris A.J.
      • Marr J.D.
      • Shiller S.M.
      • Susskind B.M.
      • et al.
      Antibody-mediated rejection as a contributor to previously unexplained early liver allograft loss.
      • Lee B.T.
      • Horwich B.H.
      • Chopra S.
      • Ahearn A.
      • Han H.H.
      Checkpoint inhibitor-induced rejection of a liver allograft: a combination of acute T cell-mediated and antibody-mediated rejection.
      Figure thumbnail gr1
      Fig. 1Representative images of lesions seen in acute AMR and C4d staining (different patient samples).
      (A) Acute AMR with endothelial cell hypertrophy (black arrows) accompanied by endothelialitis. Eosinophils are also prominent. (H&E original magnification, 400x). (B) Acute AMR with mixed portal inflammation. There is prominent eosinophilia in the infiltrate and portal vein endothelialitis is noted (black arrows). (H&E original magnification, 400x). (C) Plasma cell-rich rejection. Note the infiltrate in the portal tract consisting mainly of plasma cells. (H&E original magnification, 400x). (D) C4d immuno-stain shows positive staining in peri-sinusoidal areas. (C4d immune-stain peroxidase-anti-peroxidase original magnification, 400x). (E) C4d immune-stain shows positive staining in endothelial cells lining the portal vein (left upper corner) and septal venules (black arrows). (C4d immune-stain peroxidase-anti-peroxidase original magnification, 400x). (F) C4d immune-stain shows strong positive staining in the portal stromal tissue with sparing of the blood vessels. (C4d immune-stain peroxidase-anti-peroxidase original magnification, 100x). (G) C4d immune-stain shows positive staining in portal venules and in the peribiliary plexus. (C4d immune-stain peroxidase-anti-peroxidase original magnification, 100x). AMR, antibody-mediated rejection.
      While the Banff Working Group definition requires all 4 criteria for a definite diagnosis of acute AMR, cases of suspected AMR may not have all 4 criteria present concurrently, especially given the ubiquitous presentation with TCMR. Recent reports from the KT literature have identified histologic presentations of AMR in the absence of HLA-DSAs
      • Crespo M.
      • Redondo D.
      • Butler C.
      • Gimeno J.
      • Garcia C.
      • Perez M.J.
      • et al.
      Antibody-mediated rejection with and without HLA donor-specific antibodies in kidney-transplantation.
      ,
      • Filippone E.J.
      • Farber J.L.
      Histologic antibody-mediated kidney allograft rejection in the absence of donor specific HLA antibodies.
      or C4d-negative AMR
      • Orandi B.J.
      • Alachkar N.
      • Kraus E.S.
      • Naqvi F.
      • Lonze B.E.
      • Lees L.
      • et al.
      Presentation and outcomes of C4d-negative antibody-mediated rejection after kidney transplantation.
      ; this has yet to be described in liver AMR but certainly contributes to the elusiveness of this entity. Most cases of suspected acute AMR will present with persistent allograft dysfunction despite potent corticosteroid therapy (including steroid recycle). Subsequent DSA analysis will likely identify positivity of either HLA class I or II with mean fluorescence intensity (MFI) of ≥3,000–5,000. Histologic criteria indicative of acute AMR range from, at minimum, endothelial cell hypertrophy of portal microvasculature with focal (10–50% of portal tracts) C4d immunostaining, to severe manifestations with marked microvasculitis with disruption or interstitial haemorrhage with any positive C4d immunostaining.
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      Cases of acute liver AMR can present as refractory thrombocytopenia, hyperbilirubinaemia, low serum complement levels in the setting of graft injury and high-titre alloantibodies
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      (Table 1). In the setting of de novo DSA formation, late-onset acute AMR can develop 6 months or more after LT.
      • Del Bello A.
      • Congy-Jolivet N.
      • Muscari F.
      • Lavayssiere L.
      • Esposito L.
      • Cardeau-Desangles I.
      • et al.
      Prevalence, incidence and risk factors for donor-specific anti-HLA antibodies in maintenance liver transplant patients.
      Table 1Clinicopathological manifestations and definitions of antibody-mediated rejection.
      Clinical manifestationsPathological manifestations
      Consider serum evaluation for donor-specific antibodies and liver biopsy with staining for C4d.
      Definition
      Adapted from Demetris et al. 2016 Banff Working Group on Liver Allograft Pathology guidelines.
      Acute AMR
      • Rapid allograft failure, haemorrhagic necrosis
      • Graft injury with refractory thrombocytopenia, hyperbilirubinaemia, low serum complement levels
      • Elevated transaminases despite optimal donor liver quality
      • Portal microvascular endothelial cell hypertrophy
      • Microvasculitis/capillaritis with monocytes, eosinophils and/or neutrophils
      • Portal/peri-portal oedema
      • Microvascular injury involving central veins
      • Fibrin deposition, red blood cells seen in sub-sinusoidal regions
      • Cholestasis, ductular reaction
      • Histology (see next column)
      • Elevated DSA
      • Diffuse C4d deposition of microvasculature in ABO-compatible tissues, or portal stroma in ABO-incompatible tissues
      • Exclusion of other liver diseases or complications that can cause similar patterns of injury
      Chronic AMR
      • Can be associated with graft injury and/or advanced fibrosis without clear aetiology, sometimes in the setting of cyclosporine use or HCV infection
      • Development of portal hypertension after transplantation
      • Abnormal liver tests during immunosuppression weaning
      • Mild portal/peri-portal/peri-venular inflammation
      • Mild interface hepatitis
      • Dense portal fibrosis with collagenisation
      • Obliterative portal venopathy
      • “V” lesions
      • Periductal fibrosis, ductopenia
      • Compatible histology (see next column)
      • Positive DSA within 3 months of biopsy
      • Focal C4d positivity (>10% portal tracts)
      • Exclusion of other causes of graft injury
      AMR, antibody-mediated rejection.
      Consider serum evaluation for donor-specific antibodies and liver biopsy with staining for C4d.
      Adapted from Demetris et al. 2016 Banff Working Group on Liver Allograft Pathology guidelines.

      Chronic antibody-mediated rejection

      The true prevalence of chronic AMR is unknown, as most patients can have normal liver tests despite histologic evidence of allograft injury.
      • Feng S.
      • Bucuvalas J.C.
      • Demetris A.J.
      • Burrell B.E.
      • Spain K.M.
      • Kanaparthi S.
      • et al.
      Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants.
      ,
      • O'Leary J.G.
      • Cai J.
      • Freeman R.
      • Banuelos N.
      • Hart B.
      • Johnson M.
      • et al.
      Proposed diagnostic criteria for chronic antibody-mediated rejection in liver allografts.
      Due to the dearth of specific biochemical and histologic features, the recognition and true incidence of chronic AMR may be underappreciated. The pathophysiologic mechanism of chronic AMR in all organ allografts is not precisely known because of a lack of animal models and difficulties with in vitro modelling. Mechanisms include antibody-mediated effector pathways and antibody-mediated complement activation resulting in inflammation and fibrosis.
      • Valenzuela N.M.
      • Reed E.F.
      Antibody-mediated rejection across solid organ transplants: manifestations, mechanisms, and therapies.
      Additionally, non-inflammatory fibrosis has been noted in the presence of DSAs, possibly related to hepatic stellate cell activation,
      • Miyagawa-Hayashino A.
      • Yoshizawa A.
      • Uchida Y.
      • Egawa H.
      • Yurugi K.
      • Masuda S.
      • et al.
      Progressive graft fibrosis and donor-specific human leukocyte antigen antibodies in pediatric late liver allografts.
      along with other contributors such as transplant vasculopathy with intimal hyperplasia
      • Valenzuela N.M.
      • Reed E.F.
      Antibody-mediated rejection across solid organ transplants: manifestations, mechanisms, and therapies.
      and endothelial apoptosis.
      • Narayanan K.
      • Jaramillo A.
      • Phelan D.L.
      • Mohanakumar T.
      Pre-exposure to sub-saturating concentrations of HLA class I antibodies confers resistance to endothelial cells against antibody complement-mediated lysis by regulating Bad through the phosphatidylinositol 3-kinase/Akt pathway.
      Clinical manifestations of chronic liver AMR are non-specific and may be quiescent (Table 1).
      A consensus definition of chronic AMR has not been definitively established as studies continue to explore the spectrum of chronic AMR.
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      The criteria for probable chronic AMR include the following
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      : i) Compatible histology (see section below); ii) Positivity for DSAs within 3 months of biopsy; iii) Focal C4d positivity (>10% portal tracts), although the diffuse C4d uptake seen in acute AMR is uncommon in chronic AMR; iv) Reasonable exclusion of other liver insults. Suspected cases of chronic AMR are likely to present with histologic features of dense (keloid-like) portal and peri-sinusoidal fibrosis of the allograft, non-specific portal and peri-venular inflammation, and obliterative portal venopathy.
      Despite definitions for AMR by the Banff Working Group, clinicians and pathologists should be cognisant of over-diagnosis and be aware of the limitations in using these criteria in practice. On many occasions, AMR is associated with concomitant TCMR and/or chronic ductopenic rejection (CDR). Additionally, severe TCMR in itself may be associated with elevated DSAs and C4d positivity, as can severe plasma cell-rich rejection.
      • Schluckebier D.
      • Cousin V.L.
      • Petit L.-M.
      • Belli D.
      • Wildhaber B.
      • Rougemont A.-L.
      • et al.
      Preformed and de novo DSA are associated with T-cell-mediated rejection in pediatric liver transplant recipients requiring clinically indicated liver biopsy.
      • Castillo-Rama M.
      • Sebagh M.
      • Sasatomi E.
      • Randhawa P.
      • Isse K.
      • Salgarkar A.D.
      • et al.
      Plasma cell hepatitis" in liver allografts: identification and characterization of an IgG4-rich cohort.
      • Musat A.I.
      • Agni R.M.
      • Wai P.Y.
      • Pirsch J.D.
      • Lorentzen D.F.
      • Powell A.
      • et al.
      The significance of donor-specific HLA antibodies in rejection and ductopenia development in ABO compatible liver transplantation.
      Conventional immunosuppressive treatment of these cases may decrease C4d positivity and DSA titre, thus making the formal diagnosis of AMR more difficult to establish.

      Plasma cell-rich rejection

      Plasma cell-rich rejection (also known as “plasma cell hepatitis” or “de novo autoimmune hepatitis”) appears to be a manifestation of allograft rejection
      • Fiel M.I.
      • Agarwal K.
      • Stanca C.
      • Elhajj N.
      • Kontorinis N.
      • Thung S.N.
      • et al.
      Posttransplant plasma cell hepatitis (de novo autoimmune hepatitis) is a variant of rejection and may lead to a negative outcome in patients with hepatitis C virus.
      (Fig. 1C) and has been associated with C4d staining of portal capillaries.
      • Trivedi A.
      • Schiano T.
      • Ward S.
      • Thung S.
      • Fiel I.
      • Levitsky J.
      C4d is present in portal venules in plasma cell hepatitis and may also be used as a predictor for its development.
      However, it remains unclear if this form of rejection is an atypical presentation of acute/chronic AMR or is a separate entity. In these cases, clinicians should consider evaluating DSAs and C4d to look for underlying AMR. Some patients diagnosed with plasma cell-rich rejection may have a rapid progression of fibrosis culminating in allograft cirrhosis in the presence or absence of concomitant CDR, and they may be resistant to optimisation of immunosuppressive treatment,
      • Fiel M.I.
      • Agarwal K.
      • Stanca C.
      • Elhajj N.
      • Kontorinis N.
      • Thung S.N.
      • et al.
      Posttransplant plasma cell hepatitis (de novo autoimmune hepatitis) is a variant of rejection and may lead to a negative outcome in patients with hepatitis C virus.
      both features seen in AMR.
      Diagnosis of liver AMR must include a thorough investigation of donor-specific antibodies and characteristic histology. Patients who present with severe allograft injury should have DSA checked given the possibility of AMR.

      C4d immunostaining

      A key criterion for the diagnosis of AMR in all organ transplants is C4d staining of tissue. Previously, C4d staining was only possible by immunofluorescence of frozen tissue specimens. Practically, this can be difficult due to issues with preservation along with non-specific background staining. A recent study has confirmed the feasibility of utilising formalin-fixed paraffin-embedded (FFPE) liver tissue to detect C4d for liver AMR.
      • Neil D.A.H.
      • Bellamy C.O.
      • Smith M.
      • Haga H.
      • Zen Y.
      • Sebagh M.
      • et al.
      Global quality assessment of liver allograft C4d staining during acute antibody-mediated rejection in formalin-fixed, paraffin-embedded tissue.
      If assessing C4d deposition, the following structural compartments should be assessed: portal vein, portal capillary, portal stroma, sinusoidal and central vein endothelium. C4d deposition has a portal vein and capillary predominance in acute AMR
      • Neil D.A.H.
      • Bellamy C.O.
      • Smith M.
      • Haga H.
      • Zen Y.
      • Sebagh M.
      • et al.
      Global quality assessment of liver allograft C4d staining during acute antibody-mediated rejection in formalin-fixed, paraffin-embedded tissue.
      ,
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      ; however, a region that should be evaluated in addition to endothelial and stromal regions is the sinusoid
      • Neil D.A.H.
      • Bellamy C.O.
      • Smith M.
      • Haga H.
      • Zen Y.
      • Sebagh M.
      • et al.
      Global quality assessment of liver allograft C4d staining during acute antibody-mediated rejection in formalin-fixed, paraffin-embedded tissue.
      (Fig. 1D-F). Other common staining patterns include the portal vasculature and peribiliary capillary plexus (Fig. 1G). It is important to note that non-specific C4d staining of connective tissue can occur in the setting of sub-sinusoidal fibrosis. Further laboratory standardisation of C4d staining patterns and intensity across institutions would allow for more uniform protocols for the diagnosis and treatment of liver AMR.
      Most centres utilise rabbit polyclonal or monoclonal C4d antibodies for immunostaining of FFPE liver tissue. The most conventional C4d antibody clone used is SP91. Laboratories should validate their C4d stains with positive and negative controls (ideally C4d-positive liver allograft tissue but kidney or heart is acceptable) to ensure proper tissue processing for histopathologic assessment.
      The presence of C4d-negative AMR has been well-recognised in KT
      • Orandi B.J.
      • Alachkar N.
      • Kraus E.S.
      • Naqvi F.
      • Lonze B.E.
      • Lees L.
      • et al.
      Presentation and outcomes of C4d-negative antibody-mediated rejection after kidney transplantation.
      with molecular diagnostics of kidney allograft tissue,
      • Sis B.
      • Halloran P.F.
      Endothelial transcripts uncover a previously unknown phenotype: C4d-negative antibody-mediated rejection.
      but this has not been studied in liver AMR. Future studies focused on molecular diagnostics for liver allograft tissue will help improve our general understanding of AMR in the liver allograft.

      Donor-specific antibodies and their association with allograft function

      DSAs can be directed against either class I or II HLA or non-HLA antigens, such as angiotensin II type-1 receptor
      • Xu Q.
      • McAlister V.C.
      • Leckie S.
      • House A.A.
      • Skaro A.
      • Marotta P.
      Angiotensin II type I receptor agonistic autoantibodies are associated with poor allograft survival in liver retransplantation.
      ,
      • Wozniak L.J.
      • Hickey M.J.
      • Chan A.P.
      • Venick R.S.
      • Farmer D.G.
      • Busuttil R.W.
      • et al.
      Angiotensin II type-1 receptor antibodies are associated with active allograft dysfunction following pediatric liver transplantation.
      ; most studies of DSAs in LT focus on anti-HLA. Class II DSAs may have an impact on clinical outcomes because of the upregulation of class II antigen expression on the portal microvasculature, centrilobular endothelium, bile ducts, and hepatocytes that occurs after an initial insult to the allograft. They can be pre-formed prior to LT or develop thereafter (defined as de novo). Pre-formed class I DSAs have been observed to clear after LT, while pre-formed class II DSAs tend to persist, and have been associated with poorer outcomes.
      • Taner T.
      • Gandhi M.J.
      • Sanderson S.O.
      • Poterucha C.R.
      • De Goey S.R.
      • Stegall M.D.
      • et al.
      Prevalence, course and impact of HLA donor-specific antibodies in liver transplantation in the first year.
      ,
      • O'Leary J.G.
      • Kaneku H.
      • Jennings L.W.
      • Bañuelos N.
      • Susskind B.M.
      • Terasaki P.I.
      • et al.
      Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation.
      Clinicians should be aware of medication non-adherence or low immunosuppression levels as predisposing risk factors for de novo DSAs.
      • O'Leary J.G.
      • Samaniego M.
      • Barrio M.C.
      • Potena L.
      • Zeevi A.
      • Djamali A.
      • et al.
      The influence of immunosuppressive agents on the risk of de novo donor-specific HLA antibody production in solid organ transplant recipients.
      An MFI cut-off of 1,000-1,500 is conventionally used to characterise DSA specificity.
      • Tambur A.R.
      • Campbell P.
      • Claas F.H.
      • Feng S.
      • Gebel H.M.
      • Jackson A.M.
      • et al.
      Sensitization in transplantation: assessment of risk (STAR) 2017 working group meeting report.
      While heterogeneity exists across studies in determining the threshold limit of MFI of DSAs that is associated with clinical significance, DSAs have been implicated as a risk factor for allograft injury and failure.
      • O'Leary J.G.
      • Demetris A.J.
      • Friedman L.S.
      • Gebel H.M.
      • Halloran P.F.
      • Kirk A.D.
      • et al.
      The role of donor-specific HLA alloantibodies in liver transplantation.
      ,
      • Bouquegneau A.
      • Loheac C.
      • Aubert O.
      • Bouatou Y.
      • Viglietti D.
      • Empana J.P.
      • et al.
      Complement-activating donor-specific anti-HLA antibodies and solid organ transplant survival: a systematic review and meta-analysis.
      A recent meta-analysis of de novo DSAs utilised an MFI cut-off of 3,000 as a potential threshold associated with clinical outcomes.
      • Beyzaei Z.
      • Geramizadeh B.
      • Bagheri Z.
      • Karimzadeh S.
      • Shojazadeh A.
      De novo donor specific antibody and long-term outcome after liver transplantation: a systematic review and meta-analysis.
      It is important to note that the magnitude of MFI metrics is not the same as antibody titres, and variability exists amongst manufacturers regarding the quality of conjugation.
      • O'Leary J.G.
      • Demetris A.J.
      • Friedman L.S.
      • Gebel H.M.
      • Halloran P.F.
      • Kirk A.D.
      • et al.
      The role of donor-specific HLA alloantibodies in liver transplantation.
      ,
      • Reed E.F.
      • Rao P.
      • Zhang Z.
      • Gebel H.
      • Bray R.A.
      • Guleria I.
      • et al.
      Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA.
      A summary of studies evaluating DSA and AMR since 2016 is shown in Table 2; it is important to acknowledge that immunosuppression was not standardised across cohorts and several confounders (such as hepatitis C) were present across cohorts. Future research is anticipated from the Sensitization in Transplantation: Assessment of Risk (STAR) Working Group, which is an ongoing collaborative effort to review the quality of evidence on DSAs and transplantation.
      • Tambur A.R.
      • Campbell P.
      • Claas F.H.
      • Feng S.
      • Gebel H.M.
      • Jackson A.M.
      • et al.
      Sensitization in transplantation: assessment of risk (STAR) 2017 working group meeting report.
      ,
      • Tambur A.R.
      • Campbell P.
      • Chong A.S.
      • Feng S.
      • Ford M.L.
      • Gebel H.
      • et al.
      Sensitization in transplantation: assessment of risk (STAR) 2019 working group meeting report.
      Current data on DSAs cannot confirm a causal role in allograft injury, and we encourage a comprehensive assessment of typical causes of allograft injury despite positive DSAs.
      Table 2Published studies since 2016 evaluating donor-specific antibodies after liver transplantation.
      StudySingle vs. multi centreRetrospective vs. prospectiveAdult or paediatricN of DSA+Class I/IIPre-formed vs. de novoTechnique of DSAMFI cut-off for DSAAMR (n)Crossmatch (n)Other
      O’Leary et al. Am J Transplant 2016
      • O'Leary J.G.
      • Cai J.
      • Freeman R.
      • Banuelos N.
      • Hart B.
      • Johnson M.
      • et al.
      Proposed diagnostic criteria for chronic antibody-mediated rejection in liver allografts.
      SingleProspectiveAdult65 of 996IIn.a.SAB10,000n.a.n.a.24 patients with HCV
      O’Leary et al. Transplantation 2017
      • O'Leary J.G.
      • Smith C.
      • Cai J.
      • Hart B.
      • Jennings L.W.
      • Everly M.
      • et al.
      Chronic AMR in liver transplant: validation of the 1-year cAMR score's ability to determine long-term outcome.
      SingleProspectiveAdult230 of 850I/IIn.a.SAB1,000n.a.22111 patients with HCV
      Vandevoorde et al. Liver Transpl 2018
      • Vandevoorde K.
      • Ducreux S.
      • Bosch A.
      • Guillaud O.
      • Hervieu V.
      • Chambon-Augoyard C.
      • et al.
      Prevalence, risk factors, and impact of donor-specific alloantibodies after adult liver transplantation.
      SingleRetrospectiveAdult63 of 297I/II14 pre-formed, 59 de novoSAB1,5004n.a.2 patients with viral hepatitis
      Feng et al. Gastroenterology 2018
      • Feng S.
      • Bucuvalas J.C.
      • Demetris A.J.
      • Burrell B.E.
      • Spain K.M.
      • Kanaparthi S.
      • et al.
      Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants.
      MultiProspectivePaediatric80 of 144IIn.a.SAB, C1q2,000 SAB, 5,000 C1qScreenn.a.n.a.47 patients with LDLT, 48 of 61 with C1q+DSA
      Kubal et al. Liver Transpl 2018
      • Kubal C.A.
      • Mangus R.
      • Ekser B.
      • Mihaylov P.
      • Ceballos B.
      • Higgins N.
      • et al.
      Class II human leukocyte antigen epitope mismatch predicts de novo donor-specific antibody formation after liver transplantation.
      SingleProspectiveAdult27 of 80I/IIDe novoSAB3,000066 patients with HCV
      Guerra et al. Pediatric Transplant 2018
      • Guerra M.R.
      • Naini B.V.
      • Scapa J.V.
      • Reed E.F.
      • Busuttil R.W.
      • Cheng E.Y.
      • et al.
      Obliterative portal venopathy: a histopathologic finding associated with chronic antibody-mediated rejection in pediatric liver allografts.
      SingleRetrospectivePaediatric4I/IIn.a.SAB1,0003 of 4n.a.n.a.
      Kim et al. Clin Transplant 2018
      • Kim H.
      • Yi N.J.
      • Song E.Y.
      • Lee K.
      • Lee K.W.
      • Lee H.W.
      • et al.
      Preformed donor-specific antibodies do not affect the 1-year allograft survival in living donor liver transplantation.
      SingleProspectiveAdult15 of 61I/II15 pre-formedSAB1,000013 patients with HCV. Primary LDLT
      Kovandova et al. HLA 2018
      • Kovandova B.
      • Slavcev A.
      • Sekerkova Z.
      • Honsova E.
      • Trunecka P.
      Antibody-mediated rejection after liver transplantation-relevance of C1q and C3d-binding antibodies.
      SingleRetrospectiveAdult27 of 123I/II19 pre-formed, 8 de novoSAB1,000 for class I, 2,000 for class II121None
      Dao et al. Liver Transpl 2018
      • Dao M.
      • Habes D.
      • Taupin J.L.
      • Mussini C.
      • Redon M.J.
      • Suberbielle C.
      • et al.
      Morphological characterization of chronic antibody-mediated rejection in ABO-identical or ABO-compatible pediatric liver graft recipients.
      SingleRetrospectivePaediatric20 of 44I/IIn.a.SAB1,00013n.a.9 patients underwent LDLT
      Dumortier et al. Transpl Immunol 2019
      • Dumortier J.
      • Dedic T.
      • Erard-Poinsot D.
      • Rivet C.
      • Guillaud O.
      • Chambon-Augoyard C.
      • et al.
      Pregnancy and donor-specific HLA-antibody-mediated rejection after liver transplantation: "Liaisons dangereuses"?.
      SingleRetrospectiveAdult34 of 73I/II3 pre-formed, 31 de novoSAB1,5008n.a.All female of childbearing age
      Yazawa et al. Transpl Int 2019
      • Yazawa M.
      • Cseprekal O.
      • Helmick R.A.
      • Talwar M.
      • Balaraman V.
      • Podila P.S.B.
      • et al.
      Association between post-transplant donor specific antibodies and recipient outcomes in simultaneous liver-kidney transplant recipients: single center, cohort study.
      SingleRetrospectiveAdult12 of 85I/II4 pre-formed, 6 de novo, 2 bothSAB, C1q2,000 pre-formed, 1,000 pre-formed C1q+DSA, 1,000 de novo0 in liver, 5 in kidneyn.a.2 of 12 patients with HCV, 7 with C1q+DSA
      Jucaud et al. Hepatology 2019
      • Jucaud V.
      • Shaked A.
      • DesMarais M.
      • Sayre P.
      • Feng S.
      • Levitsky J.
      • et al.
      Prevalence and impact of de novo donor-specific antibodies during a multicenter immunosuppression withdrawal trial in adult liver transplant recipients.
      MultiProspectiveAdult24 of 40I/IIDe novoSAB1,000n.a.n.a.20 of 40 patients with HCV
      Del Bello et al. Liver Transpl 2020
      • Del Bello A.
      • Neau-Cransac M.
      • Lavayssiere L.
      • Dubois V.
      • Congy-Jolivet N.
      • Visentin J.
      • et al.
      Outcome of liver transplant patients with preformed donor-specific anti-human leukocyte antigen antibodies.
      MultiRetrospectiveAdult142 of 1788I/IIPre-formedSAB1,00010632 of 142 patients developed de novo DSA also
      Jackson et al. Am J Transplant 2020
      • Jackson A.M.
      • Kanaparthi S.
      • Burrell B.E.
      • Lucas D.P.
      • Vega R.M.
      • Demetris A.J.
      • et al.
      IgG4 donor-specific HLA antibody profile is associated with subclinical rejection in stable pediatric liver recipients.
      MultiProspectivePaediatric65 of 129IIn.a.SAB2,000n.a.n.a.Similar cohort as Feng et al.
      Wozniak et al. Transplantation 2020
      • Wozniak L.J.
      • Hickey M.J.
      • Chan A.P.
      • Venick R.S.
      • Farmer D.G.
      • Busuttil R.W.
      • et al.
      Angiotensin II type-1 receptor antibodies are associated with active allograft dysfunction following pediatric liver transplantation.
      SingleRetrospectivePaediatric42 of 70I/IIn.a.SAB1,0004n.a.AT1R non-HLA DSA assessed
      Hofer et al. Transplantation 2020
      • Hofer A.
      • Jonigk D.
      • Hartleben B.
      • Verboom M.
      • Hallensleben M.
      • Hubscher S.G.
      • et al.
      DSA are associated with more graft injury, more fibrosis, and upregulation of rejection-associated transcripts in subclinical rejection.
      SingleProspectiveBoth19 of 71IIn.a.SAB1,0000n.a.7 patients with possible chronic AMR
      AMR, antibody-mediated rejection; AT1R, angiotensin II type-1 receptor; DSA, donor-specific antibodies; LDLT, living donor liver transplantation; MFI, mean fluorescence intensity; n.a., not available; SAB, single antigen bead.
      A study by O’Leary et al. illustrated the association of DSAs and fibrosis progression after LT.
      • O'Leary J.G.
      • Kaneku H.
      • Jennings L.
      • Susskind B.M.
      • Terasaki P.I.
      • Klintmalm G.B.
      Donor-specific alloantibodies are associated with fibrosis progression after liver transplantation in hepatitis C virus-infected patients.
      There was an association noted between pre-formed class I and II DSAs and hepatic fibrosis assessed by METAVIR score, in addition to an increased risk of mortality.
      • O'Leary J.G.
      • Kaneku H.
      • Jennings L.
      • Susskind B.M.
      • Terasaki P.I.
      • Klintmalm G.B.
      Donor-specific alloantibodies are associated with fibrosis progression after liver transplantation in hepatitis C virus-infected patients.
      Notably, however, patients in this study also had recurrent HCV, which was a confounder in fibrosis progression. A more recent retrospective study of 190 patients showed a significant association of de novo DSAs with acute rejection and advanced fibrosis (a METAVIR score ≥2) at 1 year after LT (39.1% vs. 14.7% DSAs vs. no DSAs, respectively).
      • Vandevoorde K.
      • Ducreux S.
      • Bosch A.
      • Guillaud O.
      • Hervieu V.
      • Chambon-Augoyard C.
      • et al.
      Prevalence, risk factors, and impact of donor-specific alloantibodies after adult liver transplantation.
      Only 6% of patients in this study population had viral hepatitis. It is important to note that causality was not studied and whether DSAs resulted in acute rejection or vice versa is unclear. Using the 2016 Banff Criteria, 4/44 patients with de novo or persistently pre-formed DSAs were diagnosed with AMR.
      • Vandevoorde K.
      • Ducreux S.
      • Bosch A.
      • Guillaud O.
      • Hervieu V.
      • Chambon-Augoyard C.
      • et al.
      Prevalence, risk factors, and impact of donor-specific alloantibodies after adult liver transplantation.
      Feng et al. studied 157 children who underwent LT >4 years prior with normal liver tests at the time of enrollment.
      • Feng S.
      • Bucuvalas J.C.
      • Demetris A.J.
      • Burrell B.E.
      • Spain K.M.
      • Kanaparthi S.
      • et al.
      Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants.
      This study utilised the sum of DSA MFI as a value for analysis, unlike other studies of DSAs. Eighty of 144 (56%) patients had elevated class II DSAs with a MFI sum of 26,699.
      • Feng S.
      • Bucuvalas J.C.
      • Demetris A.J.
      • Burrell B.E.
      • Spain K.M.
      • Kanaparthi S.
      • et al.
      Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants.
      Those having DSA with an MFI sum >20,000 had an increased risk of fibrosis, portal inflammation, and C4d scores (including portal, capillary, sinusoidal, and total C4d staining).
      • Feng S.
      • Bucuvalas J.C.
      • Demetris A.J.
      • Burrell B.E.
      • Spain K.M.
      • Kanaparthi S.
      • et al.
      Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants.
      These patients tended to have a histopathological profile consisting of interface hepatitis and variable degrees of fibrosis despite alanine aminotransferase values <50 U/L. While suspected, it is unclear if they met the criteria for chronic AMR. Del Bello et al. recently evaluated 142 recipients across 3 French LT centres,
      • Del Bello A.
      • Neau-Cransac M.
      • Lavayssiere L.
      • Dubois V.
      • Congy-Jolivet N.
      • Visentin J.
      • et al.
      Outcome of liver transplant patients with preformed donor-specific anti-human leukocyte antigen antibodies.
      utilising the sum of DSA MFI as a value for analysis. Each patient had pre-formed DSAs at LT with a median MFI sum of 15,200.
      • Del Bello A.
      • Neau-Cransac M.
      • Lavayssiere L.
      • Dubois V.
      • Congy-Jolivet N.
      • Visentin J.
      • et al.
      Outcome of liver transplant patients with preformed donor-specific anti-human leukocyte antigen antibodies.
      Contrary to the studies above, there was no association between pre-formed DSA and fibrosis. However, a limitation of this study was that in the pre-formed DSA group more patients received induction therapy with polyclonal antibodies, anti-CD20 monoclonal antibodies (rituximab), and plasma exchange along with persistent corticosteroid use at 6 months after LT. Ten of 142 (7%) patients were diagnosed with concurrent TCMR and acute AMR.
      • Del Bello A.
      • Neau-Cransac M.
      • Lavayssiere L.
      • Dubois V.
      • Congy-Jolivet N.
      • Visentin J.
      • et al.
      Outcome of liver transplant patients with preformed donor-specific anti-human leukocyte antigen antibodies.
      The relationship between DSAs and AMR is not pathognomonic, as DSAs can be seen in TCMR, or DSAs can be elevated without clinical significance.
      • Wozniak L.J.
      • Hickey M.J.
      • Venick R.S.
      • Vargas J.H.
      • Farmer D.G.
      • Busuttil R.W.
      • et al.
      Donor-specific HLA antibodies are associated with late allograft dysfunction after pediatric liver transplantation.
      ,
      • O'Leary J.G.
      • Kaneku H.
      • Banuelos N.
      • Jennings L.W.
      • Klintmalm G.B.
      • Terasaki P.I.
      Impact of IgG3 subclass and C1q-fixing donor-specific HLA alloantibodies on rejection and survival in liver transplantation.
      More recently, improvements in the precision of HLA mismatch using eplet analysis have been suggested.
      • Wiebe C.
      • Kosmoliaptsis V.
      • Pochinco D.
      • Gibson I.W.
      • Ho J.
      • Birk P.E.
      • et al.
      HLA-DR/DQ molecular mismatch: a prognostic biomarker for primary alloimmunity.
      Small observational studies in LT have shown the relationship of eplet mismatches with class II DSAs and TCMR.
      • Kubal C.A.
      • Mangus R.
      • Ekser B.
      • Mihaylov P.
      • Ceballos B.
      • Higgins N.
      • et al.
      Class II human leukocyte antigen epitope mismatch predicts de novo donor-specific antibody formation after liver transplantation.
      • Guiral S.
      • San Segundo D.
      • Irure J.
      • Casafont F.
      • Fortea J.I.
      • Puente Á.
      • et al.
      Number of antibody verified eplet in HLA-C locus as an independent factor of T cell-mediated rejection after liver transplantation.
      • Forner D.
      • Liwski R.
      • Alwayn I.
      Human leukocyte antigen, allele, and eplet mismatches in liver transplantation; observations from a small, single center cohort.
      • Ekong U.D.
      • Antala S.
      • Bow L.
      • Sese D.
      • Morotti R.
      • Rodriguez-Davalos M.
      • et al.
      HLA, non-HLA antibodies, and eplet mismatches in pediatric liver transplantation: observations from a small, single-center cohort.
      Lastly, the correlation of serum to intra-graft DSAs and its effect on AMR in LT remains elusive. Ongoing studies analysing intra-graft DSAs will provide insights into whether intra-graft detection can be used to increase detection of AMR as a cause of allograft dysfunction and failure.
      • Neau-Cransac M.
      • Le Bail B.
      • Guidicelli G.
      • Visentin J.
      • Moreau K.
      • Quinart A.
      • et al.
      Evolution of serum and intra-graft donor-specific anti-HLA antibodies in a patient with two consecutive liver transplantations.
      ,
      • Nakamura T.
      • Shirouzu T.
      • Nakata K.
      • Yoshimura N.
      • Ushigome H.
      The role of major histocompatibility complex in organ transplantation- donor specific anti-major histocompatibility complex antibodies analysis goes to the next stage.
      Acute AMR presents with noticeable features including severe allograft injury with histology showing endothelial cell hypertrophy, microvasculitis and/or capillaritis, and central vein injury.
      Screening DSAs pre-LT may help determine which patients will require vigilant monitoring of DSA levels; however, due to a lack of large cohort studies, there is no specific recommendation per the STAR Working Group and checking for DSAs pre-LT is determined by each LT programme.
      • Tambur A.R.
      • Campbell P.
      • Claas F.H.
      • Feng S.
      • Gebel H.M.
      • Jackson A.M.
      • et al.
      Sensitization in transplantation: assessment of risk (STAR) 2017 working group meeting report.
      It is recommended that whenever a patient is found to have severe TCMR, steroid-resistant TCMR, or allograft dysfunction of unknown aetiology, DSAs should be checked, given the possibility of concomitant AMR.
      • Tambur A.R.
      • Campbell P.
      • Claas F.H.
      • Feng S.
      • Gebel H.M.
      • Jackson A.M.
      • et al.
      Sensitization in transplantation: assessment of risk (STAR) 2017 working group meeting report.

      Histology of antibody-mediated rejection

      The histopathology of AMR in LT can be extremely complex due to overlapping features seen in AMR and concomitant TCMR, particularly in smouldering injury seen in chronic AMR and late-onset TCMR.
      • Demetris A.J.
      Longterm outcome of the liver graft: the pathologist's perspective.
      AMR and TCMR (and sometimes even CDR) may all co-exist. Distinguishing the endothelialitis seen in TCMR from capillaritis and inlet venulitis seen in AMR may be extremely difficult. Even so, histological recognition of chronic AMR may be difficult to readily discern due to subtle features and less reliance on C4d staining.
      Establishing a diagnosis of chronic AMR of the liver allograft can be difficult due to the lack of specific features, resulting in less recognition by clinicians and pathologists. Additionally, potential lesions suggestive of chronic AMR can be seen concurrently with other complications of the allograft (e.g., medication-induced changes, recurrence of original disease). Histological changes suggestive of chronic AMR include unexplained mononuclear portal inflammation with interface or peri-venular necroinflammation
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      (Fig. 2A). Lesions seen in acute AMR, including endothelial cell hypertrophy and capillary dilatation, are less common.
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      There are additional suspected lesions that should lead clinicians and pathologists to consider chronic AMR as a diagnosis. O’Leary et al. assessed a cohort of elevated DSA vs. non-DSA patients, the majority of whom had HCV, with protocol liver biopsies at 1-year, finding several histologic lesions ascribable to chronic AMR.
      • O'Leary J.G.
      • Cai J.
      • Freeman R.
      • Banuelos N.
      • Hart B.
      • Johnson M.
      • et al.
      Proposed diagnostic criteria for chronic antibody-mediated rejection in liver allografts.
      These lesions included obliterative portal venopathy (OPV) and dense portal fibrosis with keloid-like features (portal tract collagenisation) (Fig. 2B). At our centre, the assessment and quantification of portal collagenisation is best appreciated with Masson trichrome staining. Extensive peri-sinusoidal and peri-venular fibrosis seen on trichrome staining may also be suggestive of chronic AMR
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      (Fig. 2C).
      Figure thumbnail gr2
      Fig. 2Representative images of lesions seen in chronic AMR.
      (A) Chronic AMR with mild interface hepatitis. There is expansion of the portal tract by a mixed inflammatory infiltrate with spillover into the peri-portal parenchyma. (H&E original magnification, 200x). (B) Chronic AMR with portal tract collagenisation and obliterative portal venopathy. Dense portal fibrosis with keloid-like features is seen along with portal venules showing luminal compromise (Trichrome stain, 100x). (C) Peri-sinusoidal and peri-venular fibrosis in centrilobular zones (Trichrome stain, 200x). (D) Nodular regenerative hyperplasia. Black arrows showing regenerative nodules (Reticulin, 200x). (E) Isolated “v” lesion with intimal hyperplasia without inflammation (H&E original magnification, 100x). (F) Senescent changes of bile ducts (yellow arrow) (H&E original magnification, 200x). AMR, antibody-mediated rejection.
      Several studies have now shown that OPV is a potential histologic lesion of chronic AMR,
      • O'Leary J.G.
      • Cai J.
      • Freeman R.
      • Banuelos N.
      • Hart B.
      • Johnson M.
      • et al.
      Proposed diagnostic criteria for chronic antibody-mediated rejection in liver allografts.
      ,
      • O'Leary J.G.
      • Smith C.
      • Cai J.
      • Hart B.
      • Jennings L.W.
      • Everly M.
      • et al.
      Chronic AMR in liver transplant: validation of the 1-year cAMR score's ability to determine long-term outcome.
      • Schiano T.D.
      • Florman S.
      • Fiel M.I.
      Recurrent idiopathic liver allograft failure.
      • Fiel M.I.
      • Schiano T.D.
      Idiopathic noncirrhotic portal hypertension.
      including in paediatric allograft recipients.
      • Guerra M.R.
      • Naini B.V.
      • Scapa J.V.
      • Reed E.F.
      • Busuttil R.W.
      • Cheng E.Y.
      • et al.
      Obliterative portal venopathy: a histopathologic finding associated with chronic antibody-mediated rejection in pediatric liver allografts.
      Nodular regenerative hyperplasia (NRH) is often seen concurrently with OPV and can be easily discerned using reticulin staining (Fig. 2D) and may also be a feature of chronic AMR.
      • Schiano T.D.
      • Florman S.
      • Fiel M.I.
      Recurrent idiopathic liver allograft failure.
      ,
      • Fiel M.I.
      • Schiano T.D.
      Idiopathic noncirrhotic portal hypertension.
      Both OPV and NRH are associated with the development of clinical portal hypertension in the liver allograft.
      • Devarbhavi H.
      • Abraham S.
      • Kamath P.S.
      Significance of nodular regenerative hyperplasia occurring de novo following liver transplantation.
      Stevenson et al. found that in patients with chronic AMR of the liver allograft, there was an isolated vascular “v” lesion, originally noted in renal allografts.
      • Stevenson H.L.
      • Prats M.M.
      • Isse K.
      • Zeevi A.
      • Avitzur Y.
      • Ng V.L.
      • et al.
      Isolated vascular "v" lesions in liver allografts: how to approach this unusual finding.
      “V” lesions are defined as inflammation, necrosis or obliterative arteriopathy in the absence of significant portal or peri-venular activity (Fig. 2E). This arteriopathy was seen in 3 patients; 1 patient was diagnosed with chronic AMR while the other 2 presumably had chronic AMR, having met 3 of the 4 criteria.
      • Stevenson H.L.
      • Prats M.M.
      • Isse K.
      • Zeevi A.
      • Avitzur Y.
      • Ng V.L.
      • et al.
      Isolated vascular "v" lesions in liver allografts: how to approach this unusual finding.
      “V” lesions are only seen in larger sized arteries (septal arteries), which are not often represented in core needle biopsies of the liver. If present, however, “v” lesions are characterised by intimal hyperplasia and arteritis. This entity is important to recognise because this might be the earliest manifestation of TCMR or an underlying chronic AMR. Biliary lesions can also be seen in chronic AMR given the potential disruption of the peri-biliary capillary plexus by DSAs. These lesions can further complicate histological assessment of AMR and delay timely diagnosis. Bile duct loss or senescence (Fig. 2F) and biliary strictures have also been associated with the presence of elevated DSAs in several studies.
      • O'Leary J.G.
      • Cai J.
      • Freeman R.
      • Banuelos N.
      • Hart B.
      • Johnson M.
      • et al.
      Proposed diagnostic criteria for chronic antibody-mediated rejection in liver allografts.
      ,
      • Iacob S.
      • Cicinnati V.R.
      • Dechêne A.
      • Lindemann M.
      • Heinemann F.M.
      • Rebmann V.
      • et al.
      Genetic, immunological and clinical risk factors for biliary strictures following liver transplantation.
      ,
      • Song S.H.
      • Kim M.S.
      • Lee J.J.
      • Ju M.K.
      • Lee J.G.
      • Lee J.
      • et al.
      Effect of donor-specific antibodies and panel reactive antibodies in living donor liver transplant recipients.

      Treatment

      No consensus exists on the optimal treatment of liver AMR; this is due to the lack of randomised controlled trials owing largely to difficulties in patient recruitment. Therefore, empirical treatment has been based on severity of graft dysfunction, DSA titres, and histologic features, and differs by centre experience. Most experiences with treatment are limited to acute AMR, and regimens are adopted from studies in KT.
      • Schinstock C.A.
      • Mannon R.B.
      • Budde K.
      • Chong A.S.
      • Haas M.
      • Knechtle S.
      • et al.
      Recommended treatment for antibody-mediated rejection after kidney transplantation: the 2019 expert consensus from the transplantion society working group.
      In addition, treatment for AMR varies widely across centres.
      • Kim P.T.
      • Demetris A.J.
      • O'Leary J.G.
      Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.
      All patients with clear cut cases of acute AMR should undergo baseline conventional rejection therapy (tacrolimus and corticosteroid-based therapy) as an initial treatment. Second-line therapy includes consideration of plasma exchange/intravenous immunoglobulin (IVIG),
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      based primarily on desensitisation protocols in ABO-incompatible transplantation and AMR treatment in the KT literature. In moderate to severe acute AMR, clinicians can consider third-line therapy with anti-CD20 agents or proteasome inhibitors although the evidence does not support this as a recommendation due to the lack of high-quality studies; this experience also derives from desensitisation protocols in ABO-incompatible transplantation and AMR treatment in the KT literature. Frequent reassessment of histology and DSAs may be necessary to help guide treatment. There is no consensus on a model for therapy in liver AMR. Some providers use a sequential approach (e.g. begin with baseline immunosuppression and assess clinical criteria or initial response before commencing plasma exchange/IVIG, etc.), while others use separate lines of therapy (e.g. begin with a proteasome inhibitor).
      A summary of recent studies evaluating treatment in acute AMR is shown in Table 3; in total, 73 patients were treated, with 24 dying and 4 re-transplanted during follow-up. As seen in the table, most cases are small, single-centre, retrospective experiences; therefore, no standard treatment modalities can be highly recommended.
      Chronic AMR presents less conspicuously, often with variable degrees of fibrosis and inflammation. Under-recognised signs of chronic AMR include obliterative portal venopathy, “v” lesions, and portal collagenisation.
      Table 3Published studies since 2016 summarising treatment of antibody-mediated rejection after liver transplantation.
      StudySingle vs. multi centreRetrospective vs. prospectiveAdult or paediatricAcute vs. chronicNType of treatmentOutcomes
      Chan KM et al. Transplantation 2011
      • Chan K.-M.
      • Lee C.-S.
      • Wu T.-J.
      • Lee C.-F.
      • Chen T.-C.
      • Lee W.-C.
      Clinical perspective of acute humoral rejection after blood type-compatible liver transplantation.
      SingleRetrospectiveBothAcute6Five of 6 patients: steroids only or steroids + rituximab + plasma exchange +/- re-transplantation

      One of 6 patients: steroids + plasma exchange + bortezomib
      Five patients dead at 11-18 days

      One alive at 180 days
      Lee CF et al. Transplant Proc 2012
      • Lee C.F.
      • Eldeen F.Z.
      • Chan K.M.
      • Wu T.H.
      • Soong R.S.
      • Wu T.J.
      • et al.
      Bortezomib is effective to treat acute humoral rejection after liver transplantation.
      SingleRetrospectiveAdultAcute9Three of 9 patients: steroids + plasma exchange + rituximab

      Six of 9 patients: steroids + plasma exchange + rituximab + bortezomib
      Six patients dead at 11-63 days

      Three alive at 18-26 months
      Paterno F Am J Transplant 2012
      • Kim P.T.
      • Demetris A.J.
      • O'Leary J.G.
      Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.
      ,
      • Paterno F.
      • Shiller M.
      • Tillery G.
      • O'Leary J.G.
      • Susskind B.
      • Trotter J.
      • et al.
      Bortezomib for acute antibody-mediated rejection in liver transplantation.
      SingleRetrospectiveAdultAcute3Bortezomib x 4 doses

      Rituximab x 4 doses, then bortezomib x 4 doses, then Bortezomib + plasma exchange x 4 cycles

      Bortezomib x 4 doses
      Death at 9 months (fibrosing cholestatic hepatitis)

      Death at 3.5 years (progressive fibrosis/liver failure)

      Re-transplantation at 1.5 years (HAT)
      Koch M et al. Transpl Int 2013
      • Koch M.
      • Gräser C.
      • Lehnhardt A.
      • Pollok J.M.
      • Kröger N.
      • Verboom M.
      • et al.
      Four-year allograft survival in a highly sensitized combined liver-kidney transplant patient despite unsuccessful anti-HLA antibody reduction with rituximab, splenectomy, and bortezomib.
      SingleRetrospectiveAdultAcute1Plasma exchange + IVIG

      Splenectomy

      Bortezomib x 4 doses + plasma exchange × 2 cycles total
      Remission
      Kim et al. Curr Opin Organ Transplant 2016
      • Kim P.T.
      • Demetris A.J.
      • O'Leary J.G.
      Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.


      Summary of Table 1 (studies prior to 2016)
      MultiRetrospectiveBothAcute11Plasma exchange, IVIG, rituximab, OKT3, polyclonal antibodies, solumedrol in different regimens4 patients dead at 40 days to 114 days

      One re-transplantation at 13 months

      Six patients alive at 72 days to 4 years
      Del Bello et al. J Gastroenterol Hepatol 2017
      • Del Bello A.
      • Danjoux M.
      • Congy-Jolivet N.
      • Lavayssiere L.
      • Esposito L.
      • Muscari F.
      • et al.
      Histological long-term outcomes from acute antibody-mediated rejection following ABO-compatible liver transplantation.
      SingleRetrospectiveAdultAcute9All 9 patients: pulse steroids 10 mg/kg daily x 3 days

      In addition:

      Seven of 9 patients: plasma exchange + rituximab
      • Three of 7 patients: + IVIG
      • Two of 7 patients: + polyclonal antibodies + IVIG
      • One of 7 patients: + polyclonal antibodies
      • One of 9 patients: rituximab
      One death at 5 months (septic shock)

      One death at 9 months (septic shock)

      One re-transplantation at 7 months

      Recurrence of AMR at 48 and 87 months
      Wozniak et al. 2017
      • Wozniak L.J.
      • Naini B.V.
      • Hickey M.J.
      • Bhattacharyya S.
      • Reed E.F.
      • Busuttil R.W.
      • et al.
      Acute antibody-mediated rejection in ABO-compatible pediatric liver transplant recipients: case series and review of the literature.
      SingleRetrospectivePaediatricAcute6Two patients with IVIG + rituximab

      One patient with IVIG

      One patient with plasma exchange + IVIG + polyclonal antibodies + bortezomib

      One patient with plasma exchange + IVIG + Rituximab

      One patient with plasma exchange + IVIG + rituximab + bortezomib + eculizumab
      One re-transplantation and death at 7.7 months (recurrent AMR)

      One death at 145 days (respiratory failure from mucous plugging)

      Four patients alive at 4.8 months to 2.7 years
      Vandevoorde et al. Liver Transpl 2018
      • Vandevoorde K.
      • Ducreux S.
      • Bosch A.
      • Guillaud O.
      • Hervieu V.
      • Chambon-Augoyard C.
      • et al.
      Prevalence, risk factors, and impact of donor-specific alloantibodies after adult liver transplantation.
      SingleRetrospectiveAdultAcute4Steroids + rituximab + plasma exchange + IVIGOne dead at 13 months

      Others with improvement at 30-70 months
      Tajima T et al. Transplant Direct 2019
      • Tajima T.
      • Hata K.
      • Okajima H.
      • Nishikori M.
      • Yasuchika K.
      • Kusakabe J.
      • et al.
      Bortezomib against refractory antibody-mediated rejection after ABO-incompatible living-donor liver transplantation: dramatic effect in acute-phase?.
      SingleRetrospectiveAdultAcute1Pre-transplant: rituximab + mycophenolic acid + tacrolimus

      Post-transplant: rituximab, then plasma exchange + IVIG + steroid pulse

      Bortezomib x 2 doses
      Remission
      Del Bello et al. Liver Transpl 2020
      • Del Bello A.
      • Neau-Cransac M.
      • Lavayssiere L.
      • Dubois V.
      • Congy-Jolivet N.
      • Visentin J.
      • et al.
      Outcome of liver transplant patients with preformed donor-specific anti-human leukocyte antigen antibodies.
      MultiRetrospectiveAdultAcute10Three of 10 patients: polyclonal antibodies + rituximab + plasma exchange

      Three of 10 patients: polyclonal antibodies

      Two of 10 patients: anti-IL2R blockers

      Two of 10 patients: none
      One death at 1 month (sepsis)

      One death at 61 months (graft failure)
      Sakamoto et al. Hepatol Res 2021
      • Sakamoto S.
      • Akamatsu N.
      • Hasegawa K.
      • Ohdan H.
      • Nakagawa K.
      • Egawa H.
      The efficacy of rituximab treatment for antibody-mediated rejection in liver transplantation: a retrospective Japanese nationwide study.
      MultiRetrospectiveBothAcute and Chronic13One of 13 patients: steroids + rituximab + polyclonal antibodies + IVIG + mTOR inhibitor

      Two: steroids + rituximab + IVIG + plasma exchange

      One: steroids + rituximab + plasma exchange

      One: rituximab + IVIG

      One: rituximab + bortezomib

      Seven of 13 patients: rituximab
      Four deaths at 12 days to 10.8 years (graft failure)

      One death at 524 days (sepsis)

      Eight patients alive at 8 to 20 years (2 patients without therapeutic efficacy of chronic AMR)
      Total N of 73 patients; 24 dead, 4 re-transplanted at follow-up. AMR, antibody-mediated rejection; HAT, hepatic artery thrombosis; IVIG, intravenous immunoglobulins.
      In chronic AMR, patients are treated with conventional rejection therapy with oral corticosteroids and tacrolimus. Literature in KT similarly recommends the use of corticosteroids, maintaining tacrolimus trough levels >5 ng/ml and supportive care.
      • Schinstock C.A.
      • Mannon R.B.
      • Budde K.
      • Chong A.S.
      • Haas M.
      • Knechtle S.
      • et al.
      Recommended treatment for antibody-mediated rejection after kidney transplantation: the 2019 expert consensus from the transplantion society working group.
      Literature on treatment of chronic liver AMR is limited due to the lack of recognition of this entity, but corticosteroid therapy has been shown to decrease the risk of de novo DSA formation
      • Del Bello A.
      • Congy-Jolivet N.
      • Muscari F.
      • Lavayssiere L.
      • Esposito L.
      • Cardeau-Desangles I.
      • et al.
      Prevalence, incidence and risk factors for donor-specific anti-HLA antibodies in maintenance liver transplant patients.
      and to decrease the risk of graft loss and patient death in simultaneous liver-kidney recipients.
      • O'Leary J.G.
      • Gebel H.M.
      • Ruiz R.
      • Bray R.A.
      • Marr J.D.
      • Zhou X.J.
      • et al.
      Class II alloantibody and mortality in simultaneous liver-kidney transplantation.
      Transplant teams should counsel patients on the importance of medication adherence, a factor crucial to decrease DSA formation. Patients without appreciable aminotransferase elevations and negligible inflammatory infiltrate on liver biopsy may be less likely to respond to anti-CD20 agents or proteasome inhibitors. Caution should be exercised when considering plasma exchange/IVIG, B cell-depleting agents, or proteasome inhibitors in cases of chronic AMR – given the lack of high-quality, well-powered, and controlled studies – in order to justify the associated risk counterbalanced with the burden of progressive fibrosis and the eventual development of CDR and/or allograft cirrhosis.
      • San Miguel J.
      • Bladé J.
      • Boccadoro M.
      • Cavenagh J.
      • Glasmacher A.
      • Jagannath S.
      • et al.
      A practical update on the use of bortezomib in the management of multiple myeloma.
      ,
      Rituximab
      LiverTox: clinical and research information on drug-induced liver injury.
      Instead, management strategies should be focused on corticosteroid/tacrolimus compliance, more frequent laboratory and histologic surveillance, and the consideration of re-transplantation. In patients with plasma cell-rich rejection, treatment with a purine analogue or mycophenolate mofetil can also be considered.
      • Stirnimann G.
      • Ebadi M.
      • Czaja A.J.
      • Montano-Loza A.J.
      Recurrent and de novo autoimmune hepatitis.

      Initial treatment of rejection

      Initial therapy should focus on treatment of concomitant TCMR given the interaction between T and B cell activity. Standard treatment includes corticosteroids with either increases in doses or bolus therapy; some facilities utilise polyclonal antibodies given their additional cytotoxic effect on B-cells
      • Kim P.T.
      • Demetris A.J.
      • O'Leary J.G.
      Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.
      The use of polyclonal antibodies, such as anti-thymocyte globulin, cannot be recommended currently given their lack of efficacy in LT
      • Boillot O.
      • Seket B.
      • Dumortier J.
      • Pittau G.
      • Boucaud C.
      • Bouffard Y.
      • et al.
      Thymoglobulin induction in liver transplant recipients with a tacrolimus, mycophenolate mofetil, and steroid immunosuppressive regimen: a five-year randomized prospective study.
      and the increased risk of infectious complications.
      • Del Bello A.
      • Neau-Cransac M.
      • Lavayssiere L.
      • Dubois V.
      • Congy-Jolivet N.
      • Visentin J.
      • et al.
      Outcome of liver transplant patients with preformed donor-specific anti-human leukocyte antigen antibodies.
      Patients on cyclosporine should be switched to an optimised tacrolimus-based regimen. Baseline immunosuppression should be strengthened with dose adjustments. There is no published data on the addition or efficacy of mTOR inhibitors.

      Plasma exchange/IVIG

      Despite a lack of strong evidence for treatment efficacy, plasma exchange and IVIG have been regarded as standard of care for acute AMR in KT.
      • Schinstock C.A.
      • Mannon R.B.
      • Budde K.
      • Chong A.S.
      • Haas M.
      • Knechtle S.
      • et al.
      Recommended treatment for antibody-mediated rejection after kidney transplantation: the 2019 expert consensus from the transplantion society working group.
      Therefore, most experiences in moderate to severe cases of AMR post-LT have similarly applied this approach.
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • O'Leary J.
      • Randhawa P.S.
      • Feng S.
      • et al.
      2016 comprehensive update of the Banff working group on liver allograft Pathology: introduction of antibody-mediated rejection.
      While plasma exchange removes IgG directly from the vascular space, several sessions are necessary to allow the antibodies in the interstitium to equilibrate for further removal. IVIG is given to replete the body of its antimicrobial IgG along with its effect on neutralising DSA formation after depletion by plasma exchange.
      • Jordan S.C.
      • Toyoda M.
      • Kahwaji J.
      • Vo A.A.
      Clinical aspects of intravenous immunoglobulin use in solid organ transplant recipients.
      High doses have been associated with a reduction in DSAs
      • Cooper J.E.
      • Gralla J.
      • Klem P.
      • Chan L.
      • Wiseman A.C.
      High dose intravenous immunoglobulin therapy for donor-specific antibodies in kidney transplant recipients with acute and chronic graft dysfunction.
      and may modulate B cell signalling and induce apoptosis. Most acute AMR treatment protocols in KT plan for plasma exchange every other day and periodic low-dose IVIG (100–500 mg/kg) for a minimum of 5 sessions.
      • Kim P.T.
      • Demetris A.J.
      • O'Leary J.G.
      Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.
      Some centres utilise high-dose IVIG (2–5 g/kg) in their protocols. While liver tests may decrease, this may not reflect durable response; therefore, optimisation of immunosuppression must continue throughout and after the use of plasma exchange and IVIG.

      Anti-CD20

      Similar to its use in KT, the use of anti-CD20 biologic agents in LT is controversial.
      • Schinstock C.A.
      • Mannon R.B.
      • Budde K.
      • Chong A.S.
      • Haas M.
      • Knechtle S.
      • et al.
      Recommended treatment for antibody-mediated rejection after kidney transplantation: the 2019 expert consensus from the transplantion society working group.
      A case series evaluated the use of rituximab in 8 patients diagnosed with acute liver AMR.
      • Del Bello A.
      • Danjoux M.
      • Congy-Jolivet N.
      • Lavayssiere L.
      • Esposito L.
      • Muscari F.
      • et al.
      Histological long-term outcomes from acute antibody-mediated rejection following ABO-compatible liver transplantation.
      Liver tests normalised in 2 patients, while DSA levels normalised in 3; C4d remained positive in 5 patients. Two patients died of sepsis, 5 and 9 months after the diagnosis of AMR. One patient required re-transplantation 7 months after diagnosis; explant histology was consistent with acute AMR. Interestingly, this study also showed evolution of histologic changes after therapy, with migration of cellular infiltrate from centrilobular to peri-portal regions and a progression of peri-portal fibrosis. A recent study from Japan evaluated 13 patients treated with rituximab for either acute (n = 4) or chronic (n = 9) liver AMR. Three of 4 patients with acute AMR died during follow-up, while there was no evidence of therapeutic efficacy in 4 of 9 patients with chronic AMR, with 2 deaths occurring during follow-up (1 sepsis, 1 graft loss).
      • Sakamoto S.
      • Akamatsu N.
      • Hasegawa K.
      • Ohdan H.
      • Nakagawa K.
      • Egawa H.
      The efficacy of rituximab treatment for antibody-mediated rejection in liver transplantation: a retrospective Japanese nationwide study.
      Additionally, in a study evaluating treatment of LT recipients with pre-formed DSAs, the use of rituximab and polyclonal antibodies was associated with an increased risk of infectious complications and death.
      • Del Bello A.
      • Neau-Cransac M.
      • Lavayssiere L.
      • Dubois V.
      • Congy-Jolivet N.
      • Visentin J.
      • et al.
      Outcome of liver transplant patients with preformed donor-specific anti-human leukocyte antigen antibodies.
      While rituximab reduces circulating B cells, the levels of peripheral IgG are relatively unchanged
      • Bingham 3rd, C.O.
      • Looney R.J.
      • Deodhar A.
      • Halsey N.
      • Greenwald M.
      • Codding C.
      • et al.
      Immunization responses in rheumatoid arthritis patients treated with rituximab: results from a controlled clinical trial.
      in contrast to the reduction in DSAs seen with IVIG, resulting in IVIG being preferred over anti-CD20 agents. The lack of high-quality studies and unproven treatment efficacy in the KT literature does not support the use of anti-CD20 agents in liver AMR.
      Treatment of AMR includes an initial conventional rejection therapy with consideration of plasmapheresis and IVIG. Treatment experience with anti-CD-20 agents and proteasome inhibitors are limited to small, mostly single-centred studies and cannot be recommended without further study.

      Proteasome inhibitors

      Proteasome inhibitors effectively target antibody-producing plasma cells within the bone marrow. A total of 13 patients have been treated with bortezomib for acute AMR after LT in case reports/series, with evidence of liver test normalisation, decreased DSA levels, and negative C4d staining in some patients. Seven patients ultimately died, 4 from allograft loss, 2 from other causes, and 1 after re-transplantation.
      • Kim P.T.
      • Demetris A.J.
      • O'Leary J.G.
      Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.
      ,
      • Chan K.-M.
      • Lee C.-S.
      • Wu T.-J.
      • Lee C.-F.
      • Chen T.-C.
      • Lee W.-C.
      Clinical perspective of acute humoral rejection after blood type-compatible liver transplantation.
      • Lee C.F.
      • Eldeen F.Z.
      • Chan K.M.
      • Wu T.H.
      • Soong R.S.
      • Wu T.J.
      • et al.
      Bortezomib is effective to treat acute humoral rejection after liver transplantation.
      • Paterno F.
      • Shiller M.
      • Tillery G.
      • O'Leary J.G.
      • Susskind B.
      • Trotter J.
      • et al.
      Bortezomib for acute antibody-mediated rejection in liver transplantation.
      • Koch M.
      • Gräser C.
      • Lehnhardt A.
      • Pollok J.M.
      • Kröger N.
      • Verboom M.
      • et al.
      Four-year allograft survival in a highly sensitized combined liver-kidney transplant patient despite unsuccessful anti-HLA antibody reduction with rituximab, splenectomy, and bortezomib.
      • Wozniak L.J.
      • Naini B.V.
      • Hickey M.J.
      • Bhattacharyya S.
      • Reed E.F.
      • Busuttil R.W.
      • et al.
      Acute antibody-mediated rejection in ABO-compatible pediatric liver transplant recipients: case series and review of the literature.
      • Tajima T.
      • Hata K.
      • Okajima H.
      • Nishikori M.
      • Yasuchika K.
      • Kusakabe J.
      • et al.
      Bortezomib against refractory antibody-mediated rejection after ABO-incompatible living-donor liver transplantation: dramatic effect in acute-phase?.
      Patients reported fatigue, nausea, and musculoskeletal pain, resulting in titration of the dosing schedules. One patient suffered from severe cytomegalovirus infection manifesting as pneumonia and pericarditis 5 months after treatment.
      • Paterno F.
      • Shiller M.
      • Tillery G.
      • O'Leary J.G.
      • Susskind B.
      • Trotter J.
      • et al.
      Bortezomib for acute antibody-mediated rejection in liver transplantation.
      Some centre protocols call for its early use after initial biopsy, while others administer it after a repeat biopsy following conventional treatment. Due to a lack of controlled studies with variations in the timing of treatment, the use of bortezomib for acute liver AMR remains controversial. Additionally, bortezomib itself carries a risk of hepatotoxicity, so clinicians must use it judiciously.
      Bortezomib
      LiverTox: clinical and research information on drug-induced liver injury.

      Re-transplantation

      Due to a lack of recommended medical therapies for AMR, centres should consider evaluation for timely re-transplantation in the setting of allograft failure. Clinicians should note that recurrence of AMR and recurrent allograft failure is possible after re-transplantation.
      • Schiano T.D.
      • Florman S.
      • Fiel M.I.
      Recurrent idiopathic liver allograft failure.
      ,
      • Duizendstra A.A.
      • Doukas M.
      • Betjes M.G.H.
      • van den Bosch T.P.P.
      • Darwish Murad S.
      • Litjens N.H.R.
      • et al.
      HLA matching and rabbit antithymocyte globulin as induction therapy to avoid multiple forms of rejection after a third liver transplantation.
      However, the risk of recurrence should not deter centres from offering re-transplantation but instead, increase awareness and allow for appropriate strategies for diagnosis and prompt management when there is concern.
      A common dilemma in patients developing allograft failure after undergoing LT decades previously is the unavailability of initial HLA profiling. In the absence of stored sera, recent advances in HLA typing using quantitative PCR assessment of 13 allograft biopsies (kidney, heart, and liver) have successfully identified HLA mismatches; the authors propose using this method to further individualise risk assessment before re-LT.
      • Berg R.
      • Nørgaard M.
      • Bruun M.T.
      • Christiansen M.
      • Koefoed-Nielsen P.
      Detecting mismatched donor HLA types from allograft biopsies - an easily applicable tool for improved individualized risk assessment.
      Anti-thymocyte globulin has shown some success for the treatment of recurrent immune-mediated graft failure in cases of HLA mismatch prior to re-LT, although further studies are needed.
      • Duizendstra A.A.
      • Doukas M.
      • Betjes M.G.H.
      • van den Bosch T.P.P.
      • Darwish Murad S.
      • Litjens N.H.R.
      • et al.
      HLA matching and rabbit antithymocyte globulin as induction therapy to avoid multiple forms of rejection after a third liver transplantation.
      A study of 79 previous LT recipients who underwent re-transplantation (39% with DSAs to second donor) showed no differences in graft or patient survival, although type of immunosuppression was not specified.
      • Xu Q.
      • Shrum B.
      • Leckie S.
      • Skaro A.
      • McAlister V.C.
      The impact of alloantibodies directed against the second donor on long-term outcomes of repeat liver transplantation.
      We do recommend assessing DSAs prior to consideration of re-transplantation in order to guide clinical management should allograft dysfunction, rejection, or other complications occur after re-transplantation. Along with standard recipient evaluation, the following should be considered: renal function and potential need for simultaneous liver-kidney transplantation, diagnosis of occult thrombophilia if thrombotic/recurrent biliary abnormalities are present, and the exclusion of hepatitis E as a possible aetiology for fibrosis progression.

      Future directions

      Open questions and research opportunities in the field are summarised in Box 1. Some centres follow DSA levels carefully and adjust immunosuppression accordingly while some centres never assess DSAs nor diagnose AMR in LT recipients. Thus, most studies in liver AMR are retrospective and single-centre experiences. Additionally, some multicentre studies often have overlapping cohorts, further increasing bias.
      • Feng S.
      • Bucuvalas J.C.
      • Demetris A.J.
      • Burrell B.E.
      • Spain K.M.
      • Kanaparthi S.
      • et al.
      Evidence of chronic allograft injury in liver biopsies from long-term pediatric recipients of liver transplants.
      ,
      • Jackson A.M.
      • Kanaparthi S.
      • Burrell B.E.
      • Lucas D.P.
      • Vega R.M.
      • Demetris A.J.
      • et al.
      IgG4 donor-specific HLA antibody profile is associated with subclinical rejection in stable pediatric liver recipients.
      Long-term outcomes of AMR, especially chronic AMR, are not well-studied, and future prospective studies are critical in understanding their natural history. Conflicting data exist on the significance of DSA formation in those in whom immunosuppression is withdrawn without graft compromise.
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      The presence of DSAs should not be used to initiate AMR-directed therapy without further investigation. Ongoing studies on immunosuppression withdrawal, especially in the paediatric population, will offer a “proof of principle’ as they provide prospective insight into the possible incidence of DSA formation and AMR development, especially given the availability of long-term follow-up
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      Prevalence and impact of de novo donor-specific antibodies during a multicenter immunosuppression withdrawal trial in adult liver transplant recipients.
      and through the performance of protocol liver biopsies. Chronic AMR in paediatric LT recipients may be easier to discern histologically given the paucity of recurrent disease which can occasionally be seen in adult LT recipients. Recently in a prospective randomised controlled trial of LT recipients undergoing immunosuppression minimisation/withdrawal, the presence of de novo class II DSAs, especially against HLA-DQ, was shown to be a predictive marker of graft rejection.
      • Jucaud V.
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      • Sayre P.
      • Feng S.
      • Levitsky J.
      • et al.
      Prevalence and impact of de novo donor-specific antibodies during a multicenter immunosuppression withdrawal trial in adult liver transplant recipients.
      Future studies are warranted to assess the role of DSA screening prior to LT.
      Outstanding questions and future research opportunities in antibody-mediated rejection after liver transplantation..
      AMR, antibody-mediated rejection; DSAs, donor-specific antibodies; HLA, human leucocyte antigen.
      Earlier and improved recognition of AMR is necessary to improve allograft outcomes in LT given the lack of proven therapies. Still, challenges remain regarding the pathologic recognition of AMR, especially in late allograft dysfunction, with the intensification of immunosuppression carrying a risk in those with long-term co-morbidities (e.g., malignancy, renal failure, cardiovascular disease). Ongoing studies on biomarkers of tolerance and rejection, in addition to genetic expression profiling, hold promise for the diagnosis of liver AMR and may help guide appropriate immunosuppression to extend both allograft and overall patient survival.
      • Celaj S.
      • Levitsky J.
      Profiling the liver graft.
      A recent study utilising molecular analysis of gene expression in biopsies of LT recipients was able to correlate rejection-associated gene transcripts with histologic TCMR, although its utility in liver AMR remains unknown.
      • Madill-Thomsen K.
      • Abouljoud M.
      • Bhati C.
      • Ciszek M.
      • Durlik M.
      • Feng S.
      • et al.
      The molecular diagnosis of rejection in liver transplant biopsies: first results of the INTERLIVER study.
      It is possible that progressive fibrosis after LT in paediatric recipients may reflect ongoing silent AMR.
      • Winters A.C.
      • Mittal R.
      • Schiano T.D.
      A review of the use of transient elastography in the assessment of fibrosis and steatosis in the post-liver transplant patient.
      Normal liver tests in the setting of progressive inflammation and fibrosis may indicate the need for protocol liver biopsies combined with routine DSA and C4d analysis, especially in patients with medication non-adherence, persistently low levels of immunosuppression, or in candidates for immunosuppression withdrawal. While acute AMR has received increasing visibility because of its overt and precipitous course towards allograft failure, the indolence of chronic AMR may confound its true impact on allograft health. Many such patients may ultimately develop concomitant CDR
      • Musat A.I.
      • Agni R.M.
      • Wai P.Y.
      • Pirsch J.D.
      • Lorentzen D.F.
      • Powell A.
      • et al.
      The significance of donor-specific HLA antibodies in rejection and ductopenia development in ABO compatible liver transplantation.
      ,
      • Shimizu A.
      • Ishii E.
      • Kuwahara N.
      • Arai T.
      • Kanzaki G.
      • Higo S.
      • et al.
      Chronic antibody-mediated responses may mediate chronic rejection in rat orthotopic liver transplantation.
      while the fibrosis of chronic AMR can progress to allograft cirrhosis.
      • Miyagawa-Hayashino A.
      • Yoshizawa A.
      • Uchida Y.
      • Egawa H.
      • Yurugi K.
      • Masuda S.
      • et al.
      Progressive graft fibrosis and donor-specific human leukocyte antigen antibodies in pediatric late liver allografts.
      ,
      • Rhu J.
      • Ha S.Y.
      • Lee S.
      • Kim J.M.
      • Choi G.S.
      • Joh J.W.
      • et al.
      Risk factors of silent allograft fibrosis 10 years post-pediatric liver transplantation.
      It is unclear if the fibrosis is potentially reversible after an increase in immunosuppression. Studies should also focus on non-invasive measures of fibrosis, such as transient elastography, for the early identification of patients with advanced allograft fibrosis and subclinical chronic AMR, especially in the context of elevated serum DSAs.
      • Winters A.C.
      • Mittal R.
      • Schiano T.D.
      A review of the use of transient elastography in the assessment of fibrosis and steatosis in the post-liver transplant patient.
      Clinicians should consider the monitoring of DSAs in at-risk patients (i.e., those with severe TCMR, steroid-resistant TCMR, unexplained allograft dysfunction). Patients with elevated DSAs need to be counselled on strict medication adherence while clinicians can maintain tighter immunosuppression and consider liver biopsy to evaluate disease activity and fibrosis stage. Future studies will need to investigate if early intervention in patients with elevated DSAs will improve long-term allograft health and decrease the need for re-transplantation. The escalation of immunosuppression due to elevated DSAs without evidence of histologic injury is controversial, and providers must exercise caution due to the potential negative impacts of intensified immunosuppression on overall patient health given long-term risks of malignancy and cardiovascular/renal injury. This dilemma must be assessed on a patient-by-patient basis with close monitoring of those who require higher doses of immunosuppression for graft survival.

      Abbreviations

      AMR, antibody-mediated rejection; CDR, chronic ductopenic rejection; DSA, donor-specific antibodies; FFPE, formalin-fixed paraffin-embedded; HLA, human leukocyte antigen; KT, kidney transplantation; LT, liver transplantation; MFI, mean fluorescence intensity; NRH, nodular regenerative hyperplasia; OPV, obliterative portal venopathy; TCMR, T cell-mediated rejection.

      Financial support

      The authors received no financial support to produce this manuscript.

      Authors’ contributions

      Lee: study concept and design, initial writing of the manuscript. Approved the final draft submitted. Fiel: study concept and design, figures, revision of the manuscript. Approved the final draft submitted. Schiano: study concept and design, revision of the manuscript. Approved the final draft submitted.

      Conflict of interest

      There are no conflicts of interest (including personal or financial) to declare from all of the authors. Writing Assistance: None. There is no shared data present in this manuscript.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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