Transplantation Reviews 29 (2015) 16–22

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The clinical relevance of alloantibody in liver transplantation C.K. Burghuber a, b, T.K. Roberts c, S.J. Knechtle a,⁎ a b c

Dept. of Surgery, Emory Transplant Center, Emory University School of Medicine, 101 Woodruff Circle, 5105 WMB, Atlanta, GA, USA Dept. of Surgery, Division of Transplantation, Medical University of Vienna, Währinger Gürtel 18–20, 1090 Vienna, Austria Dept. of Pathology and Laboratory Medicine, Emory University School of Medicine, 1364 Clifton Road NE, Atlanta, GA, USA

a b s t r a c t The transplanted liver appears resistant to antibody-mediated injury compared to other transplanted organs such as kidney or heart. However, a growing number of reports suggest that alloantibody to the liver is associated with poorer outcomes. The data surrounding this field are unclear, and their interpretation remains controversial. Mechanistically, there is not a clear explanation for the liver's resistance to antibody-mediated injury, and the pathological criteria for antibody-mediated rejection (AMR) remain ill-defined. Furthermore, treatment of AMR is non-uniform. The field would benefit from better outcome data based on measurement of antibody at the time of transplantation and at the time of rejection. Consensus opinion regarding antibody and the liver might emerge with better standardization of antibody measurement and pathological definition of AMR. © 2014 Published by Elsevier Inc.

The clinical meaning of alloantibodies in liver transplantation (LT) is much contested and difficult to elucidate. Despite studies in the past claiming that their role is negligible, a growing number of reports state the opposite. Even though only a small number of patients seem to be affected by antibody-mediated rejection in liver transplantation, the consequences of antibody-mediated injury in the context of other insults are becoming apparent. The interest in this topic is growing in parallel to antibody-related literature found for other solid organs. Since patients awaiting liver transplantation usually are very ill and denying a patient an organ for wrong reasons can be fatal, crossmatch results should be irrelevant without convincing data that a positive crossmatch impacts outcome. Nevertheless, monitoring of antibody after LT is advocated whenever a clinical consequence is likely. In this review we want to summarize the published data, the current knowledge and give a brief account on what seems to be important in a clinical context. As ABO-incompatibility is only approached in infants or in emergency situations and living donor scenarios under protocols similar to ABO-incompatible kidney transplants, this review focus mainly on antibody against HLA antigens.

1. Hyperacute and acute antibody-mediated rejection Hyperacute rejection in human allo-transplantation refers to immediate organ rejection in recipients harboring antibodies directed against epitopes on a transplanted graft. In most cases the binding of

⁎ Corresponding author at: Emory Transplant Center, Emory University School of Medicine, 101 Woodruff Circle, 5105 WMB, Atlanta, GA, USA. Tel.: +1 404 712 9910; fax: +1 404 727 3660. E-mail address: [email protected] (S.J. Knechtle). http://dx.doi.org/10.1016/j.trre.2014.06.001 0955-470X/© 2014 Published by Elsevier Inc.

preformed ABO- or donor-specific HLA-antibodies (DSA) and following complement activation at the time of graft reperfusion leads to immediate grave injury and consequent failure of the graft within 24 hours. Although a common occurrence in the early years of solid organ transplantation, hyperacute rejection rarely occurs nowadays. Avoidance or careful preparation for cases with ABO-incompatibility and the implementation of prospective DSA screening and crossmatch testing substantially reduced the incidence of hyperacute rejection following the work of Kissmeyer-Nielsen [1] and Terasaki/Patel [2–4]. Acute antibody-mediated rejection (AMR) is rarely diagnosed in LT. The basic work on its histologic features was done in animal studies [5,6] and subsequently has been followed up in human examples [7–11], but clinical presentation can be ambiguous and difficult to discern from other possible reasons for graft dysfunction. As AMR often coincides with cellular rejection patterns we will use the term acute rejection (AR) for mixed or non-specified episodes whereas AMR and acute cellular rejection (ACR) will be contrasted. The liver seems to be very resistant to acute antibody-mediated injury. Proposed explanations for this include the dual blood supply, the parenchyma's regenerative capability, an enormous potential for antibody-absorption in the large vascular bed with abundant expression of Major Histocompatibility Complex (MHC) class I antigens, the absorptive ability of Kupffer-cells [12], release of soluble MHC-antigens [13,14] and the very unique variety of resident (antigen-presenting) cells with predominant inclination to IL10-/ PD-L1-related, immunomodulatory responses (reviewed in [15]). Interesting in this context is the experimental demonstration of higher rejection rates in sensitized rats receiving reduced-sized liver grafts, probably by reason of less absorptive capacity than whole grafts [16] and higher expression of MHC class II antigens during regenerative processes after graft splitting.

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Hyperacute rejection has been experienced in rats [5,17], pigs [18], rhesus monkeys [6], and under some circumstances in humans [19,20]. The human examples show a slower process than in heart or kidney, leading to histological changes and clinical consequences, primarily graft loss, but the appearance is rare and difficult to achieve even in animal experiments. 2. Before transplantation – preformed antibody prior to liver transplantation (For the techniques of antibody-testing we refer to [21].) 2.1. HLA-mismatch HLA-mismatch has been found to significantly impact graft survival in renal transplantation, but appears to be less relevant to liver transplantation. Two international, multi-center studies concluded that HLA-matching did not impact outcomes [22,23]. 2.2. Panel-reactive antibodies (PRA) Before the development of solid-phase immunobinding assays (SPA), detection of HLA antibodies was predicated on determining the percentage of a panel of lymphocytes, with known HLA types, which was reactive with a patient's serum. For the most part, early studies using PRA as a measure of pre-sensitization found no effect on LT outcomes [24–26]. 2.3. Complement-dependent cytotoxicity crossmatch (CDCXM) The seminal work by Terasaki and Patel [2–4] demonstrated that antibodies detected by CDCXM were strongly associated with early antibody-mediated rejection and allograft loss in renal transplantation. Therefore, most early studies in the liver also concentrated on comparing CDCXM positive vs. negative patients. However, multiple reports in both animal models and patients transplanted across ABOblood groups and/or a positive CDCXM suggested that the liver was relatively resistant to hyperacute rejection [27–29]. It was noted that only minimal changes could be seen in liver specimens of these patients. These findings supported disregarding blood group disparities and cytotoxic alloantibodies in patients urgently needing liver replacement. A retrospective study including first, second, and third transplant recipients treated with cyclosporine showed no difference in 2-year graft survival between patients with positive (n = 62) or negative (n = 371) CDCXM results [24]. Furthermore, in a report of five simultaneous liver/kidney transplants, two of the recipients had a strongly positive CDCXM before surgery, which reverted to negative hours after liver perfusion and the function of the kidney grafts was maintained. This leads to the widespread perception that the liver allograft cleared anti-HLA antibody. A subsequent, retrospective study with a large cohort found higher graft failure rates in the first 6 months in CDCXM-positive patients, but the group was highly variable [30]. A study in a more homogenous cohort, comparing 25 CDCXM-positive patients with 50 matched CDCXM-negative controls, found significant deterioration of graft and patient survival in T-cell CDCXM-positive individuals, in whom the re-transplantation rate was 4 fold. For all of these studies, the standard NIH-technique for CDCXM had been used [31]. In 1996, the same group published a study using the Amos-modified technique, which has an added series of wash steps just before the addition of complement to eliminate anticomplementary factors and increase the sensitivity of the crossmatch, to identify 130 T-cell CDCXM-positive patients. This study partially confirmed their previous findings. Overall graft survival was not different at 2 years; however, a positive CDCXM was associated with a higher failure rate within the first 6–12 months. Multivariate analysis revealed that donor-age, gender, prior liver transplant, UNOS status 4

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(high risk), ischemic time, indication for transplantation, and primary immunosuppressant were independent risk factors. A positive crossmatch was only significant in the subgroup analysis of early graft losses [32]. Hathaway et al. studied this topic prospectively in 207 patients. The NIH-T- and B-cell-CDCXM was positive in 24 patients. Patient and graft survival were similar at 3 months and 1 year, but at 1 month there was a significant decrease in graft survival in the CDCXM positive group [33]. However, the difference was due to 3 early graft losses (compared to one in the CDCXM negative group), 2 of which were caused by hepatic artery thrombosis. This complication, in particular, has frequently been associated with rejection [34], but this correlation seems far from proven [1,35]. The findings of this prospective study were in accordance with previous retrospective studies. Neumann et al. studied 45 patients with a positive T-cell CDCXM (Amos technique) from a total of 793 patients. In this cohort graft survival was comparable at 1, 2, and 5 years; acute and steroidresistant rejection rates were equally distributed. Of 18 patients that underwent combined liver and kidney transplantation (CLKT) five had a positive CDCXM without hyperacute rejection or graft loss; all CLKT-patients reverted to a negative crossmatch during the immediate postoperative phase [2–4,36]. Finally Ruiz et al. retrospectively reviewed 2723 liver transplantations over a 20 year period at Baylor University [5,6,37]. Their findings were similar to previous reports, which is notable since they were using the more sensitive anti-human globulin (AHG) T-cell CDCXM technique. They examined acute and chronic rejection rates, preservation injury (PI), as well as vascular and biliary complications; however, only PI was significantly increased in CDCXM positive patients. The Collaborative Transplant Study Group summarized registry data comparing 596 T-cell-CDCXM positive with 6477 negative first-time liver transplant patients and found that graft survival was worse in the CDCXM positive group. Interestingly, the B-cell crossmatch was not a significant factor in this study [7–11,38]. Lastly the largest case series of 500 pediatric liver transplantation patients found that a positive T-cell-CDCXM was an independent risk factor for acute rejection, but not for graft or patient survival [12,39]. While these studies have, for the most part, concluded that a positive CDCXM has little impact on outcomes in liver transplant, there are a number of limitations. Firstly, the majority are retrospective studies spanning highly heterogenous patient populations using different, and now obsolete, techniques. 2.4. Flow cytometry The CDCXM was the gold standard method for alloantibody detection for over fifty years and is still widely used. However, more sensitive techniques have been developed. 2.5. Flow-cytometric crossmatch (FCXM) The FCXM does not depend on cytotoxicity, rather it detects direct binding of antibody to lymphocytes. For this reason, the sensitivity is greatly increased; however, the specificity is problematic as potentially clinical irrelevant antibodies, including autoantibodies, will also be detected. One of the first studies using FCXM in liver transplant retrospectively tested 96 cases. Of those, 10 were T-cell positive by CDCXM but 20 were positive by FCXM. A very high one-month failure rate (45% in FCXM-positive vs. 17% negative patients) was the most relevant finding [13,14,40]. In a second study, researchers found a significantly increased acute rejection rate in 13 T- and/or B-cell positive patients [15,41]. In both studies, comparison of CDCXM-results for the same patients would not have revealed significant results. A study from UCLA indicated that 1-year graft survival was significantly lower and the rate of primary non-function (PNF) higher in 76 T-cell FCXM-

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positive patients compared to 185 negative cases [16,25]. An Australian study found a higher rejection rate at 3 months and 1 year in 49 CDCXM-positive patients out of a total of 341. In this study, a positive FCXM correlated with the severity of rejection as determined by biopsy. It also discerned chronic rejection in 55 patients [5,17,42]. On an even larger scale, Scornik et al. employed FCXM in 465 liver transplant recipients and found a higher rate of steroid-resistant rejection in strongly T-cell-positive FCXM (30 × over background) patients. Overall FCXM positivity (n = 91) did not have a negative impact; paradoxically it seemed to coincide with less graft loss [18,43]. 2.6. Solid-phase methods Bead-based solid phase assays are now the foundation of HLA antibody detection in most clinical laboratories. Screening products are composed of microspheres coated with HLA proteins isolated from transformed cell lines; therefore, they express complete HLA phenotypes. These products are often considered equivalent to a PRA in that they are indicative of the degree of sensitization. The specificity of the antibodies is typically determined using single antigen beads, which are coated with only one HLA protein. These beads can precisely identify potential donor specific antibody (DSA) and allow for virtual crossmatching. A strong correlation was found between CDCXM positivity and the presence of HLA antibody detected by Luminex based screening beads [6,44]. Furthermore, a positive CDCXM combined with the presence of HLA Class II antibody was associated with shorter 1 year graft survival. Moreover, a positive bead screen alone was sufficient to predict allograft rejection. However, because they used only screening beads, it is unclear if the antibodies detected were DSA. This question was first addressed retrospectively. Sera from patients who had undergone LT across a positive FCXM were subsequently by Luminex based SABs to identify any pre-existing DSA. CDCXM and a solid-phase assay designed to detect the complement-fixing capacity of the antibodies (C1q assay) were also performed. They found that SAB results, CDCXM, and FCXM were convincingly correlated. A positive crossmatch was associated with an array of postoperative complications, but no early graft loss or mortality [19,20,45]. Taner et al. addressed this question prospectively using single antigen beads (SABs). DSA was measured on the day of liver transplant as well as 7 and 120 days post-transplant in 90 patients, who also had protocol biopsies on days 0 and 7. Twenty patients (22%) had DSA (N 2000 MFI) on the day of transplant. By day 120 post-transplant, DSA were undetectable in 17 patients (85%). Moreover, there was no difference in rate of graft loss, acute rejection, patient, or graft survival in this study. Immunohistochemical staining for C4d was more common in DSA + patients [21,46]. However, an acute rejection rate of 28% on day 7 raises the question of overreporting, as these were histologic findings in the absence of clinical graft dysfunction. Shortly thereafter, a retrospective study of 113 patients, who had pre-transplant DSA and biopsy-proved ACR within 90 days, found that low levels of DSA (≥ 300 MFI) was a risk factor for ACR. Two patients with ACR went on to develop AMR requiring antibody-depleting therapy, demonstrating the strong connection between humoral and cellular immunity in the clinical context [22,23,47]. A Baylor University study reanalyzed serum from LTpatients over 9 years at an outside laboratory in a blinded manner. With a threshold of ≥5000 MFI, they found MHC class II antibodies were an independent risk factor for early rejection and class I and/or II antibodies negatively impacted patient survival [24–26,48]. A retrospective review of patients with unexplained early graft loss revealed multiple cases with antibody-mediated injury. Saturation of SABs at a 1:27 or 1:81 dilution correlated very strongly with antibody-mediated injury. Interestingly, the percentage of SAB-positive patients was also high in those that lost their grafts due to reasons not associated with

antibody [2–4,49]. Finally, in a multivariate study confined to liver retransplants, class I DSA significantly shortened second allograft survival [27–29,50]. While these studies have provided great insight into the potential clinical relevance of DSA in liver transplantation, it is difficult to assess the mechanisms of the interaction between DSA and ACR. Nonetheless, it is clear that this interaction is critical in light of several case reports of ACR or mixed rejection, which were resistant to augmentation of conventional immunosuppression and steroids, that were resolved with antibody-depletion therapy. Furthermore, while allocation practices are unlikely to be changed based on the available data, virtual crossmatching is likely to be critical for establishing risk factors and guiding decisions about immunosuppressive regimens, induction therapy, and postoperative surveillance. 3. After transplantation – antibody persistence and de novo DSA Considering that the liver is thought to absorb antibody, the course of DSA after transplantation is an important factor. A 1992 study by Kobayashi found that 15 patients with positive postoperative CDCXM had much poorer graft survival compared to 25 patients that cleared their antibody (ie. had negative postoperative CDCXM) [24,51]. A subsequent study prospectively followed 22 consecutive patients with T-cell positive CDCXM. Ten CDCXM negative patients experiencing severe hepatocellular damage early after transplantation were included as controls. Interestingly, they found two very distinct patterns. One group (64%) apparently cleared antibodies as their positive CDCXM, which was relatively weak to begin with, became negative and stayed so after LT. This group experienced no thrombocytopenia, no increase in circulating immune complexes, and a gradual normalization of complement function within 1 week, similar to controls. Despite several cases of AR, no graft loss occurred. In contrast, in 36% of the patients with relatively high-titer (N1:32) antibodies, the CDCXM remained positive. These patients developed refractory thrombocytopenia, increased circulating immune complexes, and decreased complement activity leading to 50% graft loss [30,52]. These findings were consistent with absorption of HLA antibody by the liver but suggested that persistence of DSA posttransplant can lead to poorer outcomes. Later studies using the more sensitive FCXM seem to reach similar conclusions. In a study of 58 patients, five had a positive FCXM pretransplant, two of those persisted while another 10 patients developed a de novo positive FCXM after liver transplantation [31,53]. All of the patients with a positive post-transplant FCXM experienced at least one rejection episode within one month. In a more recent study, 19 of 197 patients were FCXM positive pretransplant. Only four did not reverse their crossmatch, three of which suffered AMR assessed by stringent criteria and lost their graft [32,54]. These studies suggest that a positive FCXM may be a risk factor for graft loss primarily due to acute rejection, but there are also data for chronic rejection. Additional to the aforementioned study [33,42], a comparison of sera from 39 liver transplant patients with biopsyproven chronic rejection demonstrated that they had a higher incidence of DSA (92% vs. 61% in 39 control patients) [34,55]. They had also developed de novo DSA more frequently, especially within the first post-transplant year. A striking finding of this study was that both persistent and de novo DSA were more often Class II, which were also associated with worse outcomes. A subsequent paper demonstrated that in the patients with DSA, those that had a predominance of IgG1 antibodies tended to have normal function while those that did develop chronic rejection had a mixed subclass population. Interestingly, the IgG3 subclass, which has the shortest half-life (7 days), was correlated with worse patient and graft survival [56,57]. A retrospective study of 749 patients found that the prevalence of de-novo DSA one year after LT was 8.1% and the vast majority of those were HLA class II antibodies. Multivariate analysis revealed that both

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graft and patient survival were lower in the DSA positive patients [58]. A prospective study of post-transplant patients had similar findings with an overall DSA prevalence of 13.1%, which were also predominantly HLA Class II antibodies. Clearance of DSA occurred in 29% of patients without any modification of therapy. DSA that persisted tended to be Class II as did de novo DSA, which developed in 9% of patients. AMR was diagnosed by biopsy in five patients, three of whom showed clinical signs of graft dysfunction and two were successfully salvaged with antibody-depleting therapy. Interestingly, the study found that the presence of DSA had no influence on patient survival [59]. Together, these findings indicate that identification of DSA pretransplant may not be a critical factor for graft and patient survival. However, if DSA persist post-transplant or are developed de novo they may lead to poorer outcomes. Particularly, since HLA Class II antigens are only present on Kupffer cells, endothelial cells, and bile ducts, clearance of Class II antibodies is limited [60]. Making matters worse, Class II antibodies are also resistant to desensitization strategies such as plasmapheresis and IVIG [61]. Nonetheless, not every patient with Class II DSA will experience rejection. Even in kidney transplantation, the exact characterization of clinically relevant antibodies has not been elucidated. Much research remains to be done before the role of DSA is adequately understood in liver transplantation.

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independent risk factor for liver graft loss and patient death [72]. Interestingly, in this study 6/20 MHC class II sensitized patients had induction therapy and these had no increased risk of liver allograft rejection. These findings are in accordance with observations published in LT alone. Together, these findings indicate that the liver is able to shield other solid organ grafts from HLA Class I DSA, but HLA Class II DSA remains problematic, negatively impacting graft and patient survival. 5. Non-HLA antibodies The clinical importance of non-HLA and non-ABO antibodies is not sufficiently clarified in kidney transplantation and in LT only very rare accounts exist. A genetic mismatch in the enzyme anti-glutathion-stransferase T1 can lead to the development of DSA, which may effectively trigger the development of de-novo immune hepatitis after LT [73]. Antibodies to biliary epithelial cells have been alleged to spark AR [74]. Anti–Major Histocompatibility Complex Class I–Related Chain A (MICA) antibodies seem to have no effect on liver grafts [75]. This area may be interesting for the future and studies are likely to parallel new developments in this area in other solid organs. 6. Injury attributed to donor specific alloantibody

4. The conundrum of combined liver–kidney transplantation

6.1. Acute antibody-mediated rejection

Combined transplantation of the liver with other solid organs of the same donor has been regarded as immunologically favorable. Combined liver–kidney transplantation (CLKT) against a positive crossmatch was accepted to be feasible and the test was then even omitted by several centers. The main reason for favorable outcomes is thought to be the absorption of DSA by the liver allograft prior to kidney perfusion. There is also speculation that liver graft-derived complement may mitigate antibody effects as seen in xenotransplants [62] and the existence of genotypic [63,64] and phenotypic [65,66] complement variants make the hypothesis feasible; however this has not been proven in allotransplants. In two early studies of combined liver and kidney transplantation (CLKT) antibodies were cleared as positive CDCXMs reverted to negative ones [24,67], but there were also two notable cases in which the liver grafts had suffered severe antibody-mediated injury and the implanted kidneys were hyperacutely rejected [19]. A case in which the liver functioned perfectly but the kidney was rapidly rejected followed [68]. Retrospective studies using CDCXM data found inferior patient and graft survival in patients with a positive crossmatch [69]. In one study using data from the Scientific Registry of Transplant Recipients (SRTR), 747 of 2484 CLKT patients were noted to have either a positive T-cell-CDCXM (n = 234) or a PRA of N10%. Multivariate analysis showed allosensitization to be an independent risk factor for patient and graft survival [70]. In retrospective work employing the Luminex based SAB assay for DSA detection, Dar et al. studied 16 CLKT patients, 6 of which had DSA and a positive FCXM. These patients were treated with IVIg before transplantation. Five had preformed MHC class II antibodies and four of them did not clear the Class II antibody post-transplant. Those four patients developed acute AMR in their renal grafts. The remaining patient without AMR was the only one that showed rapid class II antibody clearance. Five patients also had class I antibodies, which were significantly diminished within 1 month post-transplant [71]. In another study, serum samples from 86 consecutive historical CLKT patients at Baylor University were analyzed by SAB assay at Emory University in a blinded fashion. The group found 30 patients who had DSA pre-transplant and nine who developed de-novo antibodies posttransplant. Univariate analysis showed no influence of MHC class I antibodies, but class II were associated with renal AMR, liver AR, and with decreased survival of patients as well as the kidney and liver grafts. In multivariable modeling, class II antibodies remained an

Acute AMR of liver is difficult to discern in a clinical context. The coexistence of ACR, histological similarity with ischemia/reperfusion injury, and early occurrence in the first weeks after transplant make it easy to overlook. The most specific findings in cases of AMR are unexplained graft dysfunction, cholestasis without cause, refractory thrombocytopenia, decreased complement levels, and circulating immune complexes. Several case reports are available in detail [10,11,20,76]. Histology may reveal early platelet aggregation and/or margination, spotty necrosis of hepatocytes, centrilobular hepatocellular swelling, mild cholangiolar proliferation and hepatocanalicular cholestasis, microvascular injury, and inflammation [9]. On fresh frozen samples, sinusoidal C4d staining can be seen with immunofluorescence [54,77]. On formalin-fixed, paraffin-embedded tissue immunohistochemistry often shows diffuse portal microvascular and sinusoidal C4d deposition as a sign of antibody-mediated injury [78–80]. Although immunofluorescence is considered the gold standard for diagnostic C4d staining [81], specificity is sometimes problematic as it can also be seen with other pathologies [82]. Similar to kidney recipients, in ABO-incompatible cases it can be found without any clinical relevance [83]. 6.2. Biliary complications Biliary complications have always been an Achilles' heel in LT. The bile duct and its origins are predisposed to pathology for a number of reasons. Firstly, the duct's length and partly hidden trajectory can be cumbersome in dissection, especially in redo surgery. Its vasculature is primarily supplied by the hepatic artery and does not enjoy additional supply via portal branches. Small leaks of bile may not eventually mend as they generally do by an array of clotting mechanisms for blood vessels, but in contrast most likely will enlarge and discharge will increase. Despite the principally antibacterial bile, ascending colonization or infection with bile-resistant microorganisms can be encountered in liver patients, especially post papillotomy for endoscopic retrograde cholangiography (ERC). In LT the graft's bile duct comes without most of the surrounding supportive tissue, which might lessen blood supply. Finally, in some indications for LT, recipients have alterations in their duct e.g. sclerosing cholangitis. Multiple possible problems must be addressed in cases of cholestasis and/or hyperbilirubinemia. Without any proof of said etiologies,

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antibodies may contribute to injury. Matsumoto and colleagues described a markedly reduction in microvasculature per portal tract and bile duct in ACR and chronic ductopenic rejection [84]. Takaya et al. reported 3 times as many biliary complications in CDCXM positive patients compared to their negative counterparts in Pittsburgh [85]. Iacob et al. prospectively examined risk factors for biliary strictures and found ABO-compatible, but non-identical, LT to be a risk factor, suggesting immunologic contribution [86]. Fontana found 3/4 DSA positive recipients had biliary complications in Lausanne [87]. As biliary complications are relatively frequent, it is important to suspect DSA when no other reason can be found.

therapies has been reducing ischemia/reperfusion injury, but DSA usually is most deleterious when complement-binding. Eculizumab, a monoclonal antibody binding C5, has been successfully employed in sensitized kidney patients [97]. Soluble complement receptor 1 (sCR1) has been shown to inhibit ischemia/reperfusion injury and to delay onset of allograft rejection. A version engineered to temporarily insert in target cell membranes has been shown to have a 100-fold more inhibitory potential in vitro and is on trial in kidney transplantation [98].

6.3. Graft fibrosis

The role of antibody in LT remains controversial since some DSA positive patients, even those with MHC class II antibody, have successfully been weaned from all immunosuppression [99] and clear cases of antibody mediated injury are not seen in the majority of patients with detectable antibody. We deduct, especially from recent studies, that DSA can gravely injure liver allografts, and persistence or de-novo appearance after transplantation may be a problematic omen. We believe that enough evidence has been found for the negative impact of antibody on LT to recommend pre-transplant evaluation of MHC antibodies via SAB assays on a multiplex platform with the goal of measuring downstream graft injury and addressing the impact of alloantibody more definitively. FCXM would add further information. However, even in the setting of strongly positive DSA we would not advocate denying the patient the liver graft. In highly sensitized situations antibody-reducing measures should be considered. Furthermore, in cases of acute rejection that does not immediately respond to anti-cellular therapy, and graft dysfunction and/or cholestasis that cannot be clearly explained, DSA testing should be done. Even with prompt therapy using the currently available agents and means, graft survival is anything but guaranteed. Lastly, a combination of DSA testing and protocol biopsy might be useful to anticipate chronic rejection. An array of questions remain:

Several studies suggest a role for DSA in fibrosis progression. The Kyoto group showed in their adult study that DSA was related not only to ACR and C4d positivity but also to higher fibrosis scores [83]. Another Japanese group linked late onset hepatic venous outflow obstruction and the associated liver fibrosis in pediatric patients with FlowPRA positivity [88]. Miyagawa-Hayashino et al. reviewed late protocol biopsies and Luminex SAB data from 79 pediatric LT patients with good graft function. DSA was present in 48%, nearly all of which were MHC class II. Fibrosis and cirrhosis were much more prevalent in the patients with DSA [89]. Del Bello found a clear correlation of preformed and de-novo DSA with graft fibrosis [59]. Progression of fibrosis and death in HCV patients were independently associated with DSA in a study from Baylor University [90]. 7. Treatment options There is not one comparative of immunosuppressive regimens and antibody-mediated injury or DSA in LT. Some studies do include different treatment regimens, but in most this question is neither addressed nor are there specific declarations about results and power. Occasionally differences are mentioned. Cyclosporine instead of tacrolimus may be a risk factor for developing de-novo DSA [58]. There are three studies that have indicated that there may be advantages to induction with Interleukin-2-receptor antagonists (Basiliximab and Daclizumab) in LT [47,48,58]. Even less data exist for the use of lymphocyte depleting antibodies. As antibodydependent cellular cytotoxicity (ADCC) relies on NK cells, depletion would theoretically provide an interesting approach. A study retrospectively comparing FCXM positive and negative LT recipients treated with either rabbit anti-thymocyte globuline (rATG) alone or in combination with rituximab found a very low overall AR rate of only 9% in FCXM positive vs. 2% in negative patients (median follow-up 5 years). In CLKT, Dar et al. used Basiliximab in all of their patients [71] and still found MHC class II antibody persisting and causing damage to the kidney. Oddly induction therapy was not first line therapy in many CLKT studies. In treatment of AMR we rely on the experiences of kidney transplantation and case reports. Several accounts of relatively successful therapy attempts in liver graft AMR can be found in the literature. The use of IVIg and rituximab [91], IVIg, rituximab and plasmapheresis [10,92], bortezomib +/− rituximab [93] or even IVIg, rituximab, bortezomib, plasmapheresis and splenectomy in a combined sixth liver and second kidney transplant [94] have been described. These options need further evaluation before a specific therapy can be recommended. Conversion to mTOR inhibitors does not seem advisable as denovo DSA appears more often after conversion in kidney transplants [95]. In non-human primates therapies with costimulatory blockade have been shown to robustly suppress humoral responses [96], but there are as yet no conclusive data for these therapies in human LT. Approaching antibody on a different level would be interfering with complement activity. The major focus of complement inhibition

8. Conclusions

- Can immunosuppressive therapy, particularly costimulatory blockade and induction with depleting antibody, change the course of preformed or prevent the appearance of de-novo DSA? - Are there desensitization strategies that would address MHC class II antibodies more effectively? - Can we influence complement pathways to alleviate antibody injury? - Can we find a way to reliably discern antibodies that do damage from those that don't? For addressing most of these issues and to further support the role of humoral immunity in liver transplantation, randomized multicenter trials will likely be necessary. References [1] Kissmeyer-Nielsen F, Olsen S, Petersen VP, Fjeldborg O. Hyperacute rejection of kidney allografts, associated with pre-existing humoral antibodies against donor cells. Lancet 1966;2:662-5. [2] Terasaki PI, McClelland JD. Microdroplet Assay Of Human Serum Cytotoxins. Nature 1964;204:998-1000. [3] Terasaki PI, Vredevoe DL, Mickey MR. Serotyping for homotransplantation. X. Survival of 196 grafted kidneys subsequent to typing. Transplantation 1967;5: 1057-70 [Suppl]. [4] Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med 1969;280:735-9. [5] Knechtle SJ, Kolbeck PC, Tsuchimoto S, Coundouriotis A, Sanfilippo F, Bollinger RR. Hepatic transplantation into sensitized recipients. Demonstration of hyperacute rejection. Transplantation 1987;43:8-12. [6] Gubernatis G, Lauchart W, Jonker M, et al. Signs of hyperacute rejection of liver grafts in rhesus monkeys after donor-specific presensitization. Transplant Proc 1987;19:1082-3. [7] Andres GA, Ansell ID, Halgrimson CG, et al. Immunopathological studies of orthotopic human liver allografts. Lancet 1972;1:275-80. [8] Demetris AJ, Jaffe R, Tzakis A, et al. Antibody-mediated rejection of human orthotopic liver allografts. A study of liver transplantation across ABO blood group barriers. Am J Pathol 1988;132:489-502.

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