The clinical impact of donor-specific antibodies in heart transplantation

Accepted Manuscript The clinical impact of donor-specific antibodies in heart transplantation

Markus J. Barten, Uwe Schulz, Andres Beiras-Fernandez, Michael Berchtold-Herz, Udo Boeken, Jens Garbade, Stephan Hirt, Manfred Richter, Arjang Ruhpawar, Tim Sandhaus, Jan Dieter Schmitto, Felix Schönrath, Rene Schramm, Martin Schweiger, Markus Wilhelm, Andreas Zuckermann PII: DOI: Reference:

S0955-470X(17)30114-3 doi:10.1016/j.trre.2018.05.002 YTRRE 477

To appear in: Please cite this article as: Markus J. Barten, Uwe Schulz, Andres Beiras-Fernandez, Michael Berchtold-Herz, Udo Boeken, Jens Garbade, Stephan Hirt, Manfred Richter, Arjang Ruhpawar, Tim Sandhaus, Jan Dieter Schmitto, Felix Schönrath, Rene Schramm, Martin Schweiger, Markus Wilhelm, Andreas Zuckermann , The clinical impact of donorspecific antibodies in heart transplantation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ytrre(2018), doi:10.1016/j.trre.2018.05.002

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ACCEPTED MANUSCRIPT The clinical impact of donor-specific antibodies in heart transplantation

Abbreviations

antibody-mediated rejection

CAV

cardiac allograft vasculopathy

DSA

donor-specific antibodies

dnDSA

de novo DSA

HR

hazard ratio

ISHLT

International Society for Heart and Lung Transplantation

IVIG

intravenous immunoglobulin

IVUS

intravascular ultrasound

MFI

mean fluorescence intensity

NK

natural killer

rATG

rabbit antithymocyte globulin

ROC

receiver operative curves

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AMR

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ACCEPTED MANUSCRIPT Abstract

Donor-specific antibodies (DSA) are integral to the development of antibody-mediated rejection (AMR). Chronic AMR is associated with high mortality and an increased risk for cardiac allograft vasculopathy (CAV). Anti-donor HLA antibodies are present in 3–11% of patients at the time of heart transplantation (HTx), with de novo DSA (predominantly anti-

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HLA class II) developing post-transplant in 10–30% of patients. DSA are associated with

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lower graft and patient survival after HTx, with one study suggesting a three-fold increase in mortality in patients who develop de novo DSA. DSA against anti-HLA class II, notably DQ,

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are at particularly high risk for graft loss. Although detection of DSA is not a criterion for

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pathologic diagnosis of AMR, circulating DSA are found in almost all cases of AMR. MFI thresholds of ~5,000 for DSA against class I antibodies, 2,000 against class II antibodies, or

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an overall cut-off of 56,000 for any DSA, have been suggested as being predictive for AMR. There is no firm consensus on pre-transplant strategies to treat HLA antibodies, or for the elimination of antibodies after diagnosis of AMR. Minimizing the risk of dnDSA is rational but

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data on risk factors in HTx are limited. The effect of different immunosuppressive regimens is largely unexplored in HTx, but studies in kidney transplantation emphasize the importance of adherence and maintaining adequate immunosuppression. One study has suggested a

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reduced risk for dnDSA with rabbit antithymocyte globulin induction. Management of DSA pre- and post-HTx varies but typically most centers rely on a plasmapheresis or

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immunoadsorption, with or without rituximab and/or intravenous immunoglobulin. Based on the literature and a multi-center survey, an algorithm for a suggested surveillance and therapeutic strategy is provided.

Keywords: DSA, heart transplantation, donor-specific, antibody-mediated, anti-HLA

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ACCEPTED MANUSCRIPT Highlights

Pre-transplant and de novo DSA affect 3–11% and 10–30% of heart transplant patients

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DSA are integral to antibody-mediated rejection, a major cause of graft loss

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DSA are associated with higher graft loss and mortality post-heart transplant

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DSA also appears to play a contributory role in cardiac allograft vasculopathy

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Immunosuppression should minimize de novo DSA risk but data are sparse

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ACCEPTED MANUSCRIPT Introduction

Recent years have seen a substantial reduction in rates of early allograft rejection following heart transplantation (HTx). Only approximately 12% of recipients now require treatment for rejection during the first post-transplant year [1]. The frequency and severity of acute cellular rejection has been declining [2], and is now a relatively infrequent cause of death [1].

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Attention is shifting increasingly to reducing the contribution of antibody-mediated rejection

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(AMR) to graft failure, a major contributor to mortality which accounts for 35–40% of deaths before year 5 [1]. Chronic AMR (cAMR), frequently associated with accelerated cardiac

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allograft vasculopathy (CAV), plays an important role in progressive graft dysfunction [3].

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Although acute AMR during year 1 can often have a good outcome [4], late-onset cAMR diagnosed in response to graft dysfunction is associated with 50–60% mortality [4–6].

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Asymptomatic cAMR is also associated with significantly higher rates of cardiovascular mortality [7].

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As in kidney transplantation [8], donor-specific antibodies (DSA) are integral to the

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development of AMR in heart transplant recipients [5, 6, 9, 10], and there is growing evidence to show that patients with DSA are at increased risk for CAV [10–14]. Until the

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advent of solid phase assays, monitoring of anti-HLA antibodies was relatively limited and specific determination of the presence and quantity of anti-HLA class I and class II

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antibodies was not feasible. DSA surveillance using solid phase assays (SPA, e.g. Luminex) with single antigen bead (SAB) methodology, whereby recombinant HLA molecules are coated onto microbeads, allows simultaneous detection, characterization and quantification of multiple HLA antibodies, and more accurate differentiation between DSA and non-donor specific HLA antibodies [15, 16]. Pre-transplant screening for DSA and post-transplant DSA monitoring has expanded accordingly, supporting more detailed analysis of the incidence and impact of DSA in HTx and prompting more intensive investigations into therapeutic options.

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ACCEPTED MANUSCRIPT In this article, we consider techniques for assessing DSA, the current evidence base relating to the incidence, clinical impact and interventions for DSA after HTx. We present a summary of our current practice and propose an algorithm for the diagnosis and management of DSA in this setting. Non-HLA-specific antibodies, while exerting a negative effect on antibodymediated and cellular rejection after heart transplantation [17] are not discussed here.

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Assessment of DSA

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The preferred strategy for detection and monitoring of anti-HLA antibodies pre- and post-

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transplant has been discussed intensively in recent years, in terms of the choice of immunoassay, technical considerations and clinical relevance [15, 18]. This remains an

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active area of research, with many unanswered questions [19, 20]. Laboratory capabilities vary, and improved standardization and quality control has been recommended in recent

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guidelines [15]. One clear development, however, is that complement-dependent cytotoxicity (CDC) assays – which do not detect all clinically important antibodies  have been

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superseded by SPA technology due to its superior sensitivity and specificity [21]. For

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example, CDC assays can yield a false positive due to non-HLA antibodies [15]. SAB technology with SPA (Luminex), in which single purified HLA antigens are attached to

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distinguishable microspheres, determines the specificity and the relative strength of both complement-binding and noncomplement-binding HLA antibodies. SPA is the preferred

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immunoassay and is now used by most HLA laboratories, largely replacing cell-based assays. Luminex SAB testing provides more reproducible results and shows a closer correlation with clinical events in HTx recipients than flow-cytometry cross-match (FCXM) testing, which detects recipient antibodies bound to donor T and B lymphocytes [22] and which is subject to reactions caused by non-HLA antibodies [15].

Sera positive on SAB testing can be further assessed for complement-fixing antibodies by identifying C1q, one of the three subunits of complement 1, using a recently developed solid phase assay [23]. A study of endomyocardial biopsies from 44 HTx recipients with

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ACCEPTED MANUSCRIPT concurrent SAB and C1q measurement found the correlation between circulating C1q DSA and C4d on biopsy was closer than that for any DSA and C4d, although neither marker showed a significant correlation [24]. An analysis of three clinical studies by Chen et al found that the results of a complement-fixing C1q assay showed a closer correlation with AMR than IgG testing by SAB methods [23]. Other groups have also described a correlation

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between SAB-C1q reactivity and AMR in HTx patients [24]. Complement-fixing capability can

of correlation with renal and cardiac graft dysfunction [25].

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also be assessed by a C4d SPA, but appears to be less sensitive than C1q assays in terms

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In terms of cost, SPA testing using Luminex technology (SAB or 1:8 serum) is more

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expensive than CDC testing although assay costs are lower if monitoring is performed routinely, compared to testing only a single sample.

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Interpreting the clinical relevance of antibody levels can be complex. SAB was not designed to be quantifiable [26]. The level of HLA-specific antibody binding is expressed as the mean

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fluorescence intensity (MFI). MFI, despite widespread misconception, does not represent a

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titer, but instead the amount of antibody which is bound relative to the total antibody present on the beads i.e. it measures the degree of saturation [15] and should not be regarded as a quantifiable value [26]. Serum dilution is required to estimate MFI, but for example diluted

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and undiluted sera can give similar MFI readings if binding is saturated – or MFI may even

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be higher in diluted serum if there is interference by IgM in the undiluted sera [15]. So far it has not proved possible to standardize MFI between laboratories and although providing guidance, MFI cannot be regarded as a direct surrogate of antibody level.

In the current article, DSA were detected by Luminex technology unless otherwise stated.

Incidence of DSA in heart transplantation

Pre-transplant DSA

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ACCEPTED MANUSCRIPT Sensitization frequently occurs in candidates for HTx, most commonly caused by prior cardiac surgery (with extensive use of homografts), administration of blood products or use of mechanical assist devices, or due to pregnancy in female patients. Several large series have documented DSA at the time of transplant in unselected adult HTx patients using SAB technology and have reported an incidence of between 3% and 11% [17, 27–29]. The class

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of DSA in these pre-sensitized patients was not specified. In a smaller series, Topilsky and colleagues found the majority of DSA at the time of transplant (12/17 cases) to be directed

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against HLA class II antibodies [13]. In children, one small study of 25 patients found 20% to

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have DSA pre-transplant, with four of the five presensitized children having undergone

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previous surgery [30].

De novo DSA

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Relatively few studies have specifically described rates of de novo DSA (dnDSA) after HTx using SAB technology, and follow-up periods have varied considerably. In studies involving

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at least 100 adult patients followed to a mean of up to seven years post-transplant, the

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reported incidence of dnDSA has ranged from 10% to 30% [11, 17, 27, 28, 31, 32]. Evidence concerning the incidence of dnDSA over time is lacking. As in kidney transplant recipients

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[33], the greatest risk for development of dnDSA is during year 1 after HTx [31, 34], but dnDSA has first been detected for the first time as late as 19 years post-transplant [34]. De

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novo DSA against HLA class II antigens predominate (50–65%), followed by DSA against both class I and class II (18–30%) with DSA against only class I antigens being the least frequent scenario (11–20%) [11, 12, 17, 27, 31]. In children, three analyses of more than 100 children, with follow-up varying from a mean of 3.6 to 8.2 years, have reported dnDSA in 33– 43% of patients [12, 34, 35]. In these, and in a smaller series of 25 patients [30], the proportion of DSA against different classes of anti-HLA antibodies was relatively consistent: class I 20–30%, class II 47–66%, and both class I and class II 17–26% [12, 30, 34, 35]. AntiHLA-DQ specific antibodies are the predominant class II DSA after HTx [13, 17, 32, 35], as

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ACCEPTED MANUSCRIPT is the case following other types of solid organ transplant [36–38], and are associated with particularly adverse outcomes [32].

In a minority of HTx patients dnDSA are transient: one study has suggested that 15% of dnDSA do not persist, defining persistence as detection in at least two annual samples [31]. An analysis of children found 42% of dnDSA to be transient, i.e. becoming undetectable

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during a mean follow-up of 8.2 years [35]. There is some evidence that anti-HLA class II

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DSA tend to be more persistent than class I after HTx [35], but more data are required.

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DSA and survival after heart transplantation

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The evidence for significantly increased graft loss and mortality in HTx patients with DSA is convincing [11, 12, 18, 28, 31, 33]. In a series of 213 adult patients followed for a mean of 7

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years, the cumulative survival of DSA-positive patients after 5, 10, and 15 years was 89.3%, 80.3%, and 53.6% compared with 98.4% after 5 and 97.3% after 10 and 15 years for DSA-

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negative controls (only long-term survivors were screened during routine follow-up visits)

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[11]. That study did not differentiate between pre-transplant and dnDSA, but there appears to be a clear difference in outcomes in patients with pre-existing or dnDSA development.

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Impact of pre-existing versus de novo DSA

One analysis of 272 HTx recipients, 26 of whom were found to have pre-existing DSA based

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on SAB testing, showed only a short-term survival effect of class I DSA, with no short- or long-term effect of DSA overall or class II DSA [39]. Reinsmoen and colleagues assessed outcomes in 295 adult HTx patients, 14 of whom had DSA at the time of transplant which persisted post-transplant, and 32 of whom developed dnDSA [28]. At two years posttransplant the group with persistent pre-existing DSA had 100% graft survival compared to 73% survival in the dnDSA group. Clerkin et al observed comparable findings in a cohort of 221 consecutive adult patents followed for a median of 3.5 years [27]. Among those who survived to day 30, the highest survival rate was seen in patients with pre-existing DSA –

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ACCEPTED MANUSCRIPT indeed, survival was higher than those with no DSA, presumably due to HLA antibody management. The 69 patients with dnDSA, in contrast, had significantly lower survival than patients with no DSA (p=0.027) (Figure 1).

De novo DSA

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Smith et al performed a large analysis, in 243 adult HTx recipients followed for up to 13 years [31]. They observed a clear effect of dnDSA: the hazard ratio (HR) for death was

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3.067 for dnDSA (n=57) with no HLA antibodies (n=116) [31]. When a multivariate analysis

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was performed, dnDSA showed a greater impact on mortality risk than any other factor (Table 1) [31].The impact of dnDSA has also been demonstrated in children. Tran et al found

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the five-year graft survival rate to be 21% with dnDSA versus 72% in DSA-free patients in a

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cohort of 105 pediatric HTx recipients (p<0.001) [12].

The influence of different dnDSA profiles has also been examined. In the study by Clerkin

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and colleagues, the increased risk of graft loss associated with dnDSA was entirely

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attributable to the presence of class II DSA: patients without class II DSA had 100% graft survival at last follow-up [27]. Class II DQ antibodies are particularly unfavorable [35]. These findings are compatible with kidney transplantation, where patients with HLA class II alone or

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in combination with class I are at the greatest risk for AMR and graft loss [36, 40–42]. In the study by Smith et al, both complement-fixing dnDSA (HR 3.02, p=0.03) and non-

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complement-fixing dnDSA (HR 3.33, p=0.005) showed a similar association with risk for death. Of interest, a third group with non-donor-specific HLA antibodies experienced similar survival rates to the antibody-free group [31].

There are limited data on the effect of timing of dnDSA development in HTx patients. Ho and colleagues studied anti-HLA antibodies in 799 HTx recipients based on protocol biopsies performed for up to 15 years post-transplant [43]. They did not distinguish between DSA and non-donor-specific anti-HLA antibodies but, strikingly, they observed a clear difference in

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ACCEPTED MANUSCRIPT long-term survival for patients who developed DSA during year 1 (n=221) versus after year 1 (n=118). Survival rates were 52% and 40%, respectively, compared to 70% in those without any antibodies (p<0.05 and p<0.001, respectively).

DSA and development of antibody-mediated rejection

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AMR is estimated to be present in almost all late failing hearts, preceded in most cases by unrecognized long-standing subclinical AMR [9]. Diagnosis has often been challenging, but

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substantial progress has been made towards a pathology diagnosis of AMR in HTx [44].

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Based on evidence from renal transplantation, the natural history of AMR begins with the development of high-affinity alloantibodies against protein antigens in the graft, usually DSA

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[45]. The current pathology diagnostic criteria for AMR (pAMR) from the International Society for Heart and Lung Transplantation (ISHLT) do not include detection of circulating DSA,

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however, noting that DSA may not be detectable since it may be adhered to the graft.

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Using the most recent ISHLT criteria for pAMR, Loupy et al found that 14/15 patients with

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pAMR had circulating DSA at the time of graft failure compared to only 6/15 in whom their graft failed without evidence of pAMR (p<0.001) [9]. Class II DSA was immunodominant in the patients with pAMR (11/14 patients compared to class I in 3/14 patients). In another

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series, Coutance and colleagues found that all 20 patients treated for late pAMR had circulating DSA [5]. Other studies, which used solid-phase assays but applied earlier criteria

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to define AMR, detected DSA in the majority of patients at the time of AMR diagnosis (21/23 patients [43] and 6/8 patients [10]. A prospective single-center analysis of protocol endomyocardial biopsies in 113 children after HTx observed a clear relationship between the proportion of DSA-positive biopsies and the severity of pAMR, with 18%, 77% and 100% of samples graded pAMR0, pAMR1 and pAMR2 showing DSA-positivity [46]. A retrospective analysis of 66 children found DSA positivity to have a sensitivity of 92.6% for pAMR–- and noted a significant correlation between higher MFI levels and more severe pAMR [47]. It has

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ACCEPTED MANUSCRIPT been proposed that DSA monitoring by solid-phase assays may offer better prediction of pAMR than C1q-binding assays [29].

A prognostic threshold for DSA MFI values would be helpful clinically. This is challenging since DSA MFI levels vary widely. Reports have ranged from 4,500 to 26,000 in patients with late pAMR [5] and from 500 to 16,259 for pAMR with a failed graft [9], and overlap with DSA

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levels in patients with non-pAMR grafts [9]. Two analyses, both in children, have used

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receiver operative curves (ROC) to try to identify the most useful MFI cut-off points to predict pAMR. Ware et al undertook a retrospective study of 66 children, 27 of whom had at least

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one positive DSA test and 30 of whom had at least one biopsy with features of pAMR [47].

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ROC modeling showed that an MFI cut-off of 5,100 for class I antibodies (area under the curve 0.750) and 2,000 for class II antibodies (area under the curve 0.791) provided the

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maximal sensitivity and specificity for predicting pAMR grade 2 or 3 [47]. Another ROC analysis, based on 60 children, indicated that an overall MFI cut-off for any DSA of 6,000 (area under the curve 0.81) was optimal for predicting the presence of the complement

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activation fragment C4d+ [48], which is central to pAMR diagnostic criteria. Comparable

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analyses in adult HTx patients are rare, but one study reported in abstract form showed broadly similar results [49]. This was a retrospective analysis of pre-transplant serum from

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107 HTx patients, which found DSA ≥5000 MFI to be associated with higher mortality than DSA <5000 MFI over a mean follow-up of 6.6 years (HR 2.99; 95% CI 1.06-8.37). Lower cut-

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offs have also been reported, albeit infrequently. One retrospective study of 85 HTx recipients observed a pre-transplant DSA >1500 MFI to be predictive for AMR, again using Luminex technology [50]. Other authors have suggested a threshold of 2,685 MFI based on pre-transplant sera from a small cohort of lung transplant patients (n=16) [51].

In summary, detection of DSA is highly predictive for AMR–- particularly at higher levels of DSA – but circulating DSA are not universally present and AMR cannot be excluded in the absence of detectable DSA.

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ACCEPTED MANUSCRIPT DSA and cardiac allograft vasculopathy

Cardiac allograft vasculopathy is a progressive, obliterative form of intimal proliferative arteriosclerosis. It is diagnosed in 10% of adult HTx recipients by year 1, and in more than half by year 10 post-transplant [52], with a third of children developing CAV by 10 years post-transplant [53]. Although the long-term prognosis after diagnosis of CAV has improved

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[52] due to advances in percutaneous and pharmacological management, it remains a

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leading cause of late allograft loss [1, 52]. The pathogenesis of CAV is complex. Alloimmunity is the primary driver [54] but there are numerous non-immunological risk

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factors for CAV, including donor and recipient age and medical history [52].

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The contribution of AMR to CAV is increasingly being understood [55]. AMR is frequent in grafts with CAV [5, 9], and late [4] or asymptomatic AMR are associated with a higher risk for

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CAV [56]. DSA, with its integral role in AMR development, would be expected to have a relationship to risk of CAV but separately may cause direct endothelial cell damage via C4d

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complement activation and deposition or by targeting natural killer (NK) cells and

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macrophages, potentially contributing to accelerated plaque progression [57].

Where angiography or echocardiography have been used, definitions of CAV have not been

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identical, but results have usually shown higher rates of CAV in DSA-positive individuals, although conflicting data have been reported [31] (Table 2). In a retrospective analysis of

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213 patients, Kaczmarek et al showed CAV to be significantly more frequent over long-term follow-up in the subset of patients in whom DSA was detected (pre-existing or de novo), but notably the difference versus DSA-negative patients emerged only after around six years post-transplant (Figure 2) [11], reflecting the progressive nature of CAV. The interval between development of DSA and graft dysfunction due to CAV can be many months or even years, due to the slow escalation towards closure of the arterial lumen [59]. Studies with short follow-up time may thus not detect an effect of DSA.

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ACCEPTED MANUSCRIPT When pre-transplant DSA and dnDSA are analyzed separately, both have been found to be associated with an increased risk for CAV [14]. There are some data to suggest that the mean time to development of CAV was shorter with class II DSA (47 months) and, particularly, mixed class I and II DSA (25 months) than with class I DSA only (89 months) [14], but confirmatory data are lacking.

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Reducing the impact of DSA

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HLA antibody management

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Sensitized patients awaiting HTx have a high mortality rate on the waiting list due to the

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difficulty of obtaining cross-matched grafts [60], prompting the use of HLA antibody management strategies to increase the chance of transplantation. Encouragingly, there are

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reports of comparable survival rates in both sensitized adults [28] and children [60] who have a prospective positive cross-match when compared to negative-cross-match patients [61].

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Various strategies have been explored, including plasmapheresis with or without rituximab,

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intravenous immunoglobulin (IVIG) therapy [62], use of the CD52 monoclonal antibody alemtuzumab, the proteosome inhibitor bortezomib or the complement inhibitor eculizumab [63] (Figure 3). The optimal pre-transplant intervention to improve immunologic matching

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remains unclear, however, since studies of antibody-depleting strategies before HTx have generally been of low quality, involving small cohorts with short follow-up [63, 64]. Case

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reports with successful outcomes have been reported, for example using a combination of pre-transplant tacrolimus, mycophenolate mofetil, weekly immunoadsorption, IVIGs and IL-2 receptor antibody therapy [65], but comparisons of different protocols are not feasible. Innovative approaches such as antibody combination therapy [66] have also been described and merit further investigation. An important – and unanswered – question is when to undertake HLA antibody management. Since the offer of a donor heart cannot be predicted, delaying intervention

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ACCEPTED MANUSCRIPT until a graft becomes available means that only a very short protocol is possible. Preemptive intervention while on the waiting list, on the other hand, exposes the patients to an increased risk of infection and also allows time for re-emergence of anti-HLA antibodies prior to transplantation.

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Reducing the risk for de novo DSA

Given the difficulties of managing AMR, minimizing the risk of developing dnDSA is a

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rational strategy. In kidney and liver transplantation, the key risk factors for dnDSA are

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relatively well-established. The overriding concern is non-adherence to the immunosuppressive regimen [67].The extent of HLA (particularly DQ) mismatching [38, 68]

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is the leading factor, but younger age [38, 68, 69], male gender [70] and African-American race [71, 72] confer additional risk. As would be expected, sensitizing events such as

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retransplantation, pregnancy and blood transfusion or acute rejection (a marker for high immunological response) are also associated with higher rates of dnDSA. Fewer data are

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available in HTx. Godown and colleagues specifically investigated risk factors for dnDSA in a

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cohort of 121 pediatric HTx recipients, 40 of whom developed DSA by a median follow-up of 4.1 years [34]. On multivariate analysis, only mechanical circulatory support at transplant,

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African-American race and donor death from gunshot wounds showed a significant association with dnDSA. HLA matching and adherence, however, were not assessed. Other

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authors have seen an increased risk for dnDSA in presensitized patients [27] and in those with prior rejection [27] but not in male patients [11, 27] or, indeed, with increasing numbers of HLA mismatches [12]. Results are mixed concerning an association between dnDSA and prior mechanical support [11, 12, 27] or an association with younger age [11, 12, 27]. Conclusive evidence awaits larger analyses.

Data regarding an effect of the immunosuppressive regimen on risk of dnDSA after HTx are almost entirely lacking. Comparisons of HTx patients with or without dnDSA have generally not included the type or dose of immunosuppressive therapies [11, 12, 27, 34]. Recent

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ACCEPTED MANUSCRIPT reviews of evidence from kidney transplantation have concluded that inadequate immunosuppressive intensity makes development of dnDSA more likely and in high-risk patients (such as presensitized individuals), low-intensity immunosuppression – particularly early calcineurin inhibitor withdrawal without lymphocyte-depleting induction or sufficiently potent maintenance therapies – incur increased rates of dnDSA [73, 74]. Similarly,

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aggressive steroid-sparing in patients at risk for dnDSA appears inadvisable after kidney

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transplantation [74].

A rare study to assess the effect of immunosuppression on antibody production after HTx

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has been published by Rafiei et al [75]. In a retrospective analysis of 196 non-sensitized HTx

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patients, freedom from de novo antibodies at one year post-transplant in patients was significantly higher in patients given rATG induction (total dose 4.5–7.5 mg/kg) compared to

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patients without rATG (89% versus 71%, p=0.043) [75]. Freedom from DSA, specifically, was not significantly different (91% versus 88%, p=0.541). An effect on antibody production would be consistent with the findings of a retrospective study of 114 moderately sensitized

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kidney transplant recipients, which showed a lower incidence of dnDSA with rATG induction

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(mean 5 mg/kg) versus basiliximab induction (n=29) (HR 0.33, p=0.02) [76]. The mode of action of rATG is believed to include inhibition of pre-existing donor-reactive memory T-cells

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and, possibly, apoptosis of plasma calls (the source of DSA) [77].

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Studies evaluating the effect of different immunosuppressive agents and regimens on risk for dnDSA, are awaited but in the interim it may be reasonable to consider the evidence from kidney transplantation.

Managing AMR

It is currently uncertain how best to manage AMR in HTx recipients or, indeed, on whether asymptomatic AMR should be treated at all [43]. Recent consensus statements have sought to clarify the available options [43, 78], which largely overlap the therapies that are used to

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ACCEPTED MANUSCRIPT treat sensitized patients prior to transplantation. AMR is typically managed empirically using a combination of various therapies (Figure 3), with extracorporeal treatments such as plasmapheresis, plasma exchange and immunoadsorption forming the basis for intervention [79, 80]. Therapeutic plasma exchange was found in one retrospective analysis to rapidly improve graft function in HTx recipients with AMR [81]. A less common extracorporeal

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intervention after HTx is photopheresis, whereby peripheral lymphocytes are collected by apheresis and T-cell apoptosis is induced by 8-methoxypsoralen and ultraviolet light at

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multiple sessions over a couple of months. Currently, it is recommended as chronic therapy

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for AMR [80]. However, there are few data to guide protocols, patient selection or adjunctive therapies for photopheresis therapy [82]. Patients with a low level of sensitization may be

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managed with intravenous steroids and IVIG [62]. More highly sensitized HTx patients with AMR can benefit from adjunctive therapy with rituximab, which appears to improve survival

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[83, 84]. Other interventions include rabbit antithymocyte globulin (rATG) and, less frequently, bortezomib or eculizumab [85, 86] with rare instances of belatacept

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administration [87]. Single-center reports in small populations have documented highly

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variable reductions in DSA concentrations in children treated with plasma exchange therapy [88, 89] or with combined bortezomib-containing HLA antibody management regimens [89,

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90] to manage AMR after HTx.

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DSA: Current practice in heart transplantation

The ISHLT recently published a consensus document on the management of antibodies in heart transplantation [44] but the scarcity of well-designed studies in HTx examining the prognostic implications of different levels and types of DSA, optimal monitoring protocols, and the appropriate timing and type of treatment mean that management strategies still vary widely. The authors collated information on the diagnosis and management strategies for DSA at their centers, representing current practice at 15 centers in Germany, Austria and Switzerland (including one specialist pediatric center) (Table 3). The total number of HTx

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ACCEPTED MANUSCRIPT procedures at these centers from 2006 to 2016 was 3,456. The majority of centers (80%) undertake routine DSA monitoring after HTx, starting at various time points between day 0 and month 3, with a minimum of testing at months 3, 6 and 12. Monitoring continues with reduced frequency (every 312 months) after the first year. All centers measure DSA in the event of primary graft failure or graft dysfunction of unknown causes, and the majority also

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measure DSA if acute rejection or cardiac allograft vasculopathy develops (Table 3). Luminex SAB testing is used universally, with less frequent use of C1q monitoring, CDC and

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flow cytometry. Two-thirds of centers apply MFI thresholds for dnDSA when deciding

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whether or not to intervene, adopting values in the range 10003000 MFI as a cut-off for

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anti-HLA class I or anti-HLA class II.

All centers treat HLA antibodies if patients develop dnDSA with echocardiographic evidence

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of graft dysfunction or dnDSA with documented AMR, and 10/15 do so if dnDSA is present with symptoms of heart failure. All respondents stated that therapy was stopped when

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dnDSA became undetectable or below threshold levels, or when dnDSA was still present but

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echocardiography showed improved heart function or if AMR or heart failure symptoms had resolved. (Achieving undetectable dnDSA may not be possible due to high background levels of antibodies in the serum after treatment, for example with plasmapheresis.)

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Fourteen centers provided information on their approach to treating dnDSA, with immunoglobulin therapy, immunoadsorption, rATG and plasmapheresis predominating

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(Table 3). The immunosuppressive regimen is changed in response to the first detection of dnDSA in 4/15 centers, including the introduction of tacrolimus if not already administered and increasing MMF dose and everolimus exposure, with 7/13 changing the regimen if dnDSA persists after treatment of HLA antibodies (Table 3).

Given the paucity of robust data in HTx patients, no definitive protocol for DSA surveillance and treatment can be developed. However, based on the procedures employed at the authors’ centers, a provisional algorithm for the instigation of HLA reduction and

17

ACCEPTED MANUSCRIPT management of DSA in response to DSA detection and clinical findings is suggested in Figure 4.

DSA monitoring after HTx can provide a relatively early diagnostic marker to trigger intervention protocols to reduce HLA, potentially facilitating intervention before the graft becomes damaged beyond recovery. Much remains to be investigated, however. A

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multicenter study to collect serum at defined time points pre- and post-transplant in order to

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analyze DSA would be optimal, but in its absence it would be valuable to establish a DSA registry in HTx. The objectives of such a registry could include defining the time points to

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measure dnDSA, determining MFI thresholds, investigating the value of C1q monitoring and

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refining pre- and postoperative management strategies which are effective but less toxic

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than at present.

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ACCEPTED MANUSCRIPT References

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38. Kaneku H, O'Leary JG, Banuelos N, et al. De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients Am J Transplant 2013;13:1541–8. 39. Raess M, Fröhlich G, Roos M et al. Donor-specific anti-HLA antibodies detected by Luminex: predictive for short-term but not long-term survival after heart transplantation. Transplant Int 2013;26:1097–107. 40. Fidler SJ, Irish AB, Lim W, Ferrari P, Witt CS, Christiansen FT. Pre-transplant donor specific anti-HLA antibody is associated with antibody-mediated rejection, progressive graft dysfunction and patient death. Transpl Immunol 2013;28:148–53. 41. Cooper JE, Gralla J, Chan L, Wiseman AC. Clinical significance of post kidney transplant de novo DSA in otherwise stable grafts. Clin Transpl 2011:359– 64. 42. Zecher D, Bach C, Staudner C, et al. Characteristics of donor-specific antiHLA antibodies and outcome in renal transplant patients treated with a standardized induction regimen. Nephrol Dial Transplant 2017;32:730–7. 43. Ho EK, Vlad G, Vasilescu ER, et al. Pre- and posttransplantation allosensitization in heart allograft recipients: Major impact of de novo alloantibody production on allograft survival. Hum Immunol 2011;72:5–10. 44. Kobashigawa J, Colvin M, Potena L, Dragun D, Crespo-Leiro MG, Delgado JF et al. The management of antibodies in heart transplantation: An ISHLT consensus document. J Heart Lung Transplant 2018; S1053-2498(18)31292-0. 45. Loupy A, Hill GS, Jordan SC. The impact of donor-specific anti-HLA antibodies on late kidney allograft failure. Nat Rev Nephrol 2012;8:348–57. 46. Tible M, Loupy A, Vernerey D, et al. Pathologic classification of antibody-mediated rejection correlates with donor-specific antibodies and endothelial cell activation. J Heart Lung Transplant 2013;32:769–76. 47. Ware Al, Malmberg E, Delgado JC, et al. The use of circulating donor specific antibody to predict biopsy diagnosis of antibody-mediated rejection and to provide prognostic value after heart transplantation in children. J Heart Lung Transplant 2016;35:179–85. 48. Peng DM, Law YM, Kemna MS, Warner P, Nelson K, Boucek RJ. Donor-specific antibodies: can they predict C4d deposition in pediatric heart recipients? Pediatr Transplant 2013;17:429–35. 49. Dhingra R, Yu MD, Johnson M, et al. Journal of Heart and Lung Transplantation. Conference: 36th Annual Meeting and Scientific Sessions of the International Society for Heart and Lung Transplantation, ISHLT 2016. Washington, DC United States. Conference Publication: (var.pagings). 35 (4 SUPPL. 1) (pp S42), 2016. 50. Gandhi MJ, De Goey SR, Bundy K, et al. Effect of pretransplant human leukocyte antigen antibodies detected by solid-phase assays on heart transplant outcomes. Transplant Proc 2011;43:3840–6. 51. Liu C, Wetter L, Pang S, Phelan DL, Mohanakumar T, Morris GP. Cutoff values and data handling for solid-phase testing for antibodies to HLA: effects on listing unacceptable antigens for thoracic organ transplantation. Hum Immunol 2012;73:597– 604. 52. Stehlik J, Edwards LB, Kucheryavaya AY, et al. The Registry of the International Society for Heart and Lung Transplantation: 29th Official Adult Heart Transplant Report - 2012. J Heart Lung Transplant 2012;31:1052–64. 53. Aurora P, Edwards LB, Kucheryavaya AY, et al. The Registry of the International Society for Heart and Lung Transplantation: thirteenth official pediatric lung and heartlung transplantation report--2010.J Heart Lung Transplant 2010;29:1129–41. 54. Pober JS, Jane-wit D, Qin L, Tellides G. Interacting mechanisms in the pathogenesis of cardiac allograft vasculopathy. Arterioscler Thromb Vasc Biol 2014;34:1609–14. 55. Uber WE, Self SE, Van Bakel AB, Pereira NL. Acute antibody-mediated rejection following heart transplantation. Am J Transplant 2007;7:2064–74.

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56. Wu GW, Kobashigawa JA, Fishbein MC, et al. Asymptomatic antibody-mediated rejection after heart transplantation predicts poor outcomes. J Heart Lung Transplant 2009;28:417–22. 57. Wehner J, Morrell CN, Reynolds T, Rodriguez ER, Baldwin WM 3rd.Antibody and complement in transplant vasculopathy. Circ Res 2007;100:191–203. 58. Mehra MR, Crespo-Leiro MG, Dipchand A et al. International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010. J Heart Lung Transplant 2010;29:717–27. 59. Terasaki PI, Cai J. Human leukocyte antigen antibodies and chronic rejection: from association to causation. Transplantation 2008;86:377–83. 60. Feingold B, Park SY, Comer DM, et al. Outcomes after listing with a requirement for a prospective crossmatch in pediatric heart transplantation. J Heart Lung Transplant 2013;32:56–62. 61. Pollock-BarZiv SM, Hollander ND, Ngan BY, et al. Pediatric heart transplantation in human leukocyte antigen-sensitized patients: evolving management and assessment of intermediate-term outcomes in a high-risk population. Circulation 2007;116(suppl I):I172–8. 62. Jordan SC, Toyoda M, Kahwaji J, Vo AA. Clinical aspects of intravenous immunoglobulin use in solid organ transplant recipients. Am J Transplant 2011;11:196– 202. 63. Chih S, Patel J. Desensitization strategies in adult heart transplantation: Will persistence pay off? J Heart Lung Transplant 2016;35:962–72. 64. Chang DH, Kobashigawa JA. Desensitization strategies in the patient awaiting heart transplantation. Curr Opin Cardiol 2017;32:301–7. 65. Bućin D, Gustafsson R, Ekmehag B, et al. Desensitization and heart transplantation of a patient with high levels of donor-reactive anti-human leukocyte antigen antibodies. Transplantation 2010;90:1220–5. 66. Shariff H, Tanriver Y, Brown KL, et al. Intermittent antibody-based combination therapy removes alloantibodies and achieves indefinite heart transplant survival in presensitized recipients. Transplantation 2010;90:270–8. 67. Sellarés J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence. Am J Transplant 2012;12:388–99. 68. Wiebe C, Gibson IW, Blydt-Hansen TD, et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. Am J Transplant 2012;12:1157–67. 69. Del Bello A, Congy-Jolivet N, Muscari F, et al. Prevalence, incidence and risk factors for donor-specific anti-HLA antibodies in maintenance liver transplant patients. Am J Transplant 2014;14:867–75. 70. Kanter Berga J, Pallardo Mateu LM, Beltran Catalan S, et al. Donor-specific HLA antibodies: risk factors and outcomes after kidney transplantation. Transplant Proc 2011;43:2154–6. 71. DeVos JM, Gaber AO, Knight RJ, et al. Donor-specific HLA-DQ antibodies may contribute to poor graft outcome after renal transplantation. Kidney Int 2012;82:598– 604. 72. Cole RT, Gandhi J, Bray RA, et al. Racial differences in the development of de-novo donor-specific antibodies and treated antibody-mediated rejection after heart transplantation. J Heart Lung Transplant 2018;37:503–12. 73. O'Leary JG, Samaniego M, Crespo Barrio M, et al. The influence of immunosuppressive agents on the risk of de novo donor-specific HLA antibody production in solid organ transplant recipients. Transplantation 2016;100:39–53. 74. Grimbert P, Thaunat O. mTOR inhibitors and risk for chronic antibody-mediated rejection after kidney transplantation: Where are we now? Transpl Int 2017;30:647–57. 75. Rafiei M, Kittleson M, Patel J, et al. Anti-thymocyte gamma-globulin may prevent antibody production after heart transplantation. Transplant Proc 2014;46:3570–4.

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76. BrokhofMM, Sollinger HW, Hager DR, et al. Antithymocyte globulin is associated with a lower incidence of de novo donor-specific antibodies in moderately sensitized renal transplant recipients. Transplantation 2014;97:612–7. 77. Pascual J, Zuckermann A, Djamali A, Hertig A, Naesens M. Rabbit antithymocyte globulin and donor-specific antibodies in kidney transplantation - a review. Transplant Rev (Orlando) 2016;30:85–91. 78. Colvin MM, Cook JL, Chang P, et al. Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association. Circulation 2015;131:1608–39. 79. Rummler S, Barz D. Plasma exchange and immunoadsorption of patients with thoracic organ transplantation. Transfus Med Hemother 2012;39:234–40. 80. Kittleson MM, Kobashigawa J. Antibody-mediated rejection. Curr Opin Organ Transplant 2012;17:551–7. 81. Sing H, Vanlandingham S, Halverson C, et al. Therapeutic plasma exchange rapidly improves cardiac allograft function in patients with presumed antibody-mediated rejection. J Clin Apher 2014;29:316–21. 82. Barten MJ, Dieterien MT. Extraocorporeal photopheresis after heart transplantation. Immunotherapy 2014;6:927–44. 83. Ravichandran AK, Schilling JD, Novak E, Pfeifer J, Ewald GA, Joseph SM. Rituximab is associated with improved survival in cardiac allograft patients with antibody-mediated rejection: a single-center review. Clin Transplant 2013;27:961–7. 84. Aggarwal A, Pyle J, Hamilton J, Bhat G. Low-dose rituximab therapy for antibodymediated rejection in a highly sensitized heart-transplant recipient. Tex Heart Inst J 2012;39:901–5. 85. Chih S, Tinckam KJ, Ross HJ. A survey of current practice for antibody-mediated rejection in heart transplantation. Am J Transplant 2013;13:1069–74. 86. Thrush PT, Pahl E, Naftel DC, et al. A multi-institutional evaluation of antibody-mediated rejection utilizing the Pediatric Heart Transplant Study database: Incidence, therapies and outcomes. J Heart Lung Transplant 2016;35:1497–1504. 87. Enderby CY, Habib P, Patel PC, Yip DS, Orum S, Hosenpud JD. Belatacept maintenance in a heart transplant recipient. Transplantation 2014;98:e74–5. 88. Jackups R Jr, Center C, Sweet SC, Mohanakumar T, Morris GP. Measurement of donor-specific HLA antibodies following plasma exchange therapy predicts clinical outcome in pediatric heart and lung transplant recipients with antibody-mediated rejection. J Clin Apher 2013;28:301–8. 89. Morrow WR, Frazier EA, Mahle WT, et al. Rapid reduction in donor-specific anti-human leukocyte antigen antibodies and reversal of antibody-mediated rejection with bortezomib in pediatric heart transplant patients. Transplantation 2012;93:319–24. 90. Zinn MD, L'Ecuyer TJ, Fagoaga OR, Aggarwal S. Bortezomib use in a pediatric cardiac transplant center. Pediatr Transplant 2014;18:469–76. 91. Berry GJ, Angelini A, Burke MM, et al. The ISHLT working formulation for pathologic diagnosis of antibody-mediated rejection in heart transplantation: Evolution and current status (2005–2011). J Heart Lung Transplant 2011;30:601–11.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Graft loss in adult heart transplant patients with no DSA (n=141), pre-existing DSA (n=18) or de novo DSA (n=53) (Kaplan-Meier estimates) during a median follow-up of 3.5 years). (Reproduced with permission from reference 27) Figure 2. Freedom from cardiac allograft vasculopathy(CAV) in adult heart transplant recipients with DSA (n=23) or without DSA (n=190) (Reproduced with permission from reference 11)

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Figure 3. Sites of action of DSA therapies. APC, antigen-presenting cell; IVIG, intravenous immunoglobulin

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Figure 4. Suggested algorithm for the instigation of AMR therapy in response to donor specific antibodies (DSA) detection (by solid phase assay [SPA]) , MFI 15000)or heart failure symptoms (HFS) after heart transplantation. Two different clinical scenarios of DSA detection are common. Firstly, in a patient with heart failure symptoms the work-up shows DSA positivity, or, secondly, DSA positivity is detected by surveillance monitoring (SM) in an asymptomatic patient. In both cases, the following diagnostic procedures should be performed: (1) biopsy for pathological diagnosis of antibody mediated rejection (pAMR); (2) echocardiography (echo) and/or right heart catheter (rhc) for assessment of graft dysfunction (GDF). ; (3) physical examination and laboratory values (Lab) for HFS. A C1q test should be considered because C1q+DSA are accompanied with a worth outcome than C1q-DSA, and, therefore, trigger a more vigorous treatment 44.

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Treatment should be initiated if DSA is present plus at least one of the other diagnostic findings is confirmed i.e. pAMR ≥1 ISHLT [44, 91]; or GDF (echo: systolic left ventricular ejection fraction <45% or severe diastolic restriction; rhc, i.e. cardiac index <2.5 L/min/m², pulmonary capillary pressure >12mmHg). ; SPA detection of DSA with HLA class I and II MFI10003000. ; and an increase in B-natriuretic peptide (BNP) or NT-pro BNP ≥2.5 (based on the most recent laboratory value) in combination with clinical signs of HFS on physical examination.

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As Stage I of treatment, switching to a tacrolimus (TAC) regimen plus mycophenoalate mofetil (MMF) or everolimus (ERL) plus steroids, or increasing immunosuppression (e.g. higher doses) in a TAC-based regimen, is recommended. Depending on the severity of AMR or GDF with or without heart failure symptoms, it is at the discretion of the heart transplant center whether to start Stage II treatment immediately or to check the efficacy of Stage I treatment first. Stage II treatment includes different therapy options, allowing physicians to individualize intervention depending on the severity of AMR (e.g. clinical symptoms, GDF, pAMR, or the level of DSA). Although clinical data are still sparse, extracorporeal photophoresis (ECP) for a number of months may be helpful for treating chronic DSA positivity or severe cases of AMR [82]. Regardless of the treatment option selected, the efficacy of Stage II treatment should be checked. HFS, heart failure symptoms; SM, surveillance monitoring; TAC, tacrolimus; MMF, mycophenolic mofetil; ERL, everolimus; lab of HF, laboratory values of heart failure; PS, pulse steroids; PA, plasmapheresis; IA, immunadsorption; IVIG, intravenous immunoglobulin; rATG, rabbit antithymocyte globulin; RA, rituximab; BO bortezomib; EA, eculizumab

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ACCEPTED MANUSCRIPT Table 1. Multivariate analysis of the association between persistent de novo DSA (n=48) and risk of mortality in 243 adult heart transplant recipients at a single center. HLA antibody was measured annually (maximum follow-up 13 years) [31] Hazard ratio

95% CI

P value

Persistent de novo DSA

4.33

1.92, 9.76

<0.001

HLA-DR mismatch

2.33

1.08, 5.05

0.032

Donor age

1.03

1.00, 1.08

0.26

Hemodynamic compromise

0.36

1.00, 5.58

0.050

Treated rejection by year 1

0.42

1.83, 0.95

0.038

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Variable

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ACCEPTED MANUSCRIPT Table 2. Incidence of cardiac allograft vasculopathy(CAV) according to presence or absence of donor-specific antibodies (DSA) after heart transplantation in retrospective studies Study

n

DSA a incidence

CAV diagnosis

Frank et al 2014 [14]

112

Class 1: n=5 Class II: n=26 Both: n= 20

Angiography (ISHLT 2010 guidelines [58])

Topilsky et al 2013 [13]

51

Class 1: n=4 Class II: n=11

Irving et al 2015 [35]

108

Class 1: n=12 Class II: n=23 Both: n= 8

Kaczmarek et al 2008 [11]

213

Class I: n=4 Class II: d n=12 d Both: n=7

Smith et al, 2011 [31]

243

Tran et al, 2016 [12]

105

Class 1: n=8 Class II: n=37 Both: n= 12 De novo DSA: Class 1: n=8 Class II: n=28 Both: n=8

a b

b

b

d

No. patients with CAV 24

Follow-up

Incidence of CAV

Not stated

CAV grade ≥1: DSA+: 32% CAV DSA–: 13% CAV

Angiography (ISHLT 2010 guidelines [58])

43

Median 3.7 years

Angiography or post-mortem (no definition provided) Angiography (new-onset stenosis >30% or severe rarefaction of small coronary branches) Angiography (≥25% stenosis in ≥1 coronary artery) Angiography (any new-onset coronary artery stenosis or luminal irregularity)

10

Mean 8.2 years

CAV grade ≥1 at 4 c years : DSA class II: 100% CAV DSA–: 63% CAV e p=0.05 e CAV at 10 years : DSA+: 32% DSA–: 12% p=0.048 e CAV at 10 years : DSA+: 59% DSA–:33% p=0.025

Not stated

Mean 7 years

25/57 (DSA+ patients) 24

Maximum 13 years

DSA+: n=25 DSA–: not stated

Mean 3.6 years

DSA+: 36% DSA–: 13% p<0.01

T P E

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A

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N A

Additional comments

Mean time to CAV varies according to type of DSA: Class I: 89 months Class II: 47 months Both : 25 months c CAV grade ≥2 at 4 years : Class II: 58% CAV DSA–: 18% CAV

On multivariate analysis, DSA was an independent risk factor for CAV (p=0.038) On multivariate analysis, DSA was an independent risk factor for CAV (odds ratio 2.96; p=0.009)

De novo DSA not significantly associated with CAV (hazard ratio 1.06, p=0.850) On multivariate analysis, DSA was not an independent predictor for CAV

Pre-existing or de novo DSA, unless otherwise stated Children aged <18 years

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ACCEPTED MANUSCRIPT c

Too few patients with DSA Class I to assess relation with CAV Screening with ELISA assay, with solid-phase technology with single antigen beads used to identify antigen specificity in samples with HLA antibodies detected e Kaplan-Meier estimates IVUS, intravascular ultrasound All studies using single antigen beads with Luminex-based technology unless otherwise stated d

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ACCEPTED MANUSCRIPT Table 3. Diagnosis and management of DSA: key findings from a survey of 15 heart transplant centers in Germany, Austria and Switzerland in 2017. Data were not provided on all points by each center.

Routine DSA monitoring post-transplant

DSA assay types

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MFI threshold applied when considering intervention for dnDSA

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Clinically-triggered DSA monitoring post-transplant

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Initial immunosuppression in sensitized patients

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Pre-transplant HLA a antibody management

3,456 across all 15 centers during 20062016 Adult and pediatric transplant programs Mean number of procedures per center per year: 21 The most frequent interventions are:  Plasmapheresis (pre- or peri-operative) – 100%  Immunoadsorption – 53%  Immunoglobulin therapy (pre- or peri-operative) - 100%  Rituximab – 73%  Bortezomib – 33%  rATG (peri-operative) – 67% (8/10 Thymoglobulin, 2/10 Neovii) b  Tacrolimus 87% b  Cyclosporine 40%  Mycophenolic acid  100%  Everolimus – 13%  Steroids  100% Yes – 80%  6/12 start at month 1, 4/12 start at month 3, 1 at day 0 and 1 at time of listing  After month 3, monitoring is every 36 months to month 12 in all 12 centers that perform routine monitoring  7/12 centers continue monitoring after month 12 at least annually Centers measure DSA in response to the following events:  Acute rejection – 93%  Cardiac allograft vasculopathy – 67%  Primary graft failure – 100%  Graft dysfunction of unknown cause – 100% The most frequent assays are:  Luminex single-antigen bead pre- and post-transplant – 100%  C1q  pre-transplant 33%, post-transplant 20%  CDC  pre-transplant 53%, post-transplant 73%  Flow cytometry pre- and post-transplant – 20% Yes – 40% Among 4 centers which provided thresholds:  HLA DSA I threshold: 1000-1500 at 3/4 centers, 3000 at 1/4 center  HLA DSA II threshold: 1000-1500 at 3/4 centers, 3000 at 1/4 center  dnDSA alone – 60%  dnDSA + echocardiographic evidence of graft dysfunction – 100%  dnDSA + AMR – 100%  dnDSA + heart failure symptoms – 73%  No dnDSA – 60%  dnDSA normal/improved + echocardiographic evidence of improved graft function – 100%  dnDSA normal/improved + no AMR – 100%  dnDSA normal/improved + no heart failure symptoms – 100%  Immunoglobulin therapy – 79%  Rituximab – 79%  Immunoadsorption – 50%  rATG – 50% (4/7 Thymoglobulin, 3/7 Neovii))  Plasmapheresis – 43%  Extracorporeal photopheresis – 29%  Basiliximab – 14%  Bortezomib – 7%

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Heart transplant procedures

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Indicators to start dnDSA treatment

Indicators to stop dnDSA treatment

Treatment for dnDSA

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ACCEPTED MANUSCRIPT Yes – 64% Among 7 centers which provided information on specific changes:  Increase tacrolimus exposure – 2/7  Switch CsA to tacrolimus – 1/7  Switch MMF to everolimus – 3/7  rATG – 1/7 (Thymoglobulin) a Not all interventions are used in all cases b Some centers use tacrolimus or CsA, depending on individual patient profiles c 14 centers provided data d 13 centers provided data

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Immunosuppression changes n response to a,d dnDSA

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ACCEPTED MANUSCRIPT The clinical impact of donor-specific antibodies in heart transplantation Markus J. Barten, MDa, Uwe Schulz, MDb, Andres Beiras-Fernandez, MDc, Michael Berchtold-Herz, MDd, Udo Boeken, MDe, Jens Garbade, MDf, Stephan Hirt, MDg, Manfred Richter, MDh, Arjang Ruhpawar, MDi, Tim Sandhaus, MDj, Jan Dieter Schmitto, MDk, Felix Schönrath, MDl, Rene Schramm, MDm, Martin Schweiger, MDn, Markus Wilhelm, MDo,

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Andreas Zuckermann, MDp a

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University Heart Center, University Hospital Hamburg-Eppendorf, Martinistrasse 52, 20246

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Hamburg, Germany b

Clinic for Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, Ruhr-

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University Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany c

55128 Mainz, Germany d

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Department of Cardiac and Thoracic Surgery, University of Mainz, Langenbeckstrasse 1,

Department of Cardiovascular Surgery, Heart Center Freiburg University, Hugstetter

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Straße 55, 79106 Freiburg, Germany e

Düsseldorf, Germany f

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Department of Cardiovascular Surgery, Heinrich Heine University, Moorenstr. 5, 40225

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Department of Cardiac Surgery, University Hospital Leipzig, Heart Center Leipzig,

Strümpellstr. 39, 04289 Leipzig, Germany g

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Department of Cardiac and Thoracic Surgery, University of Regensburg, Franz-Josef-

Strauss-Allee 11, 93053 Regensburg, Germany h

Kerckhoff Clinic, Benekestraße 2-8, 61231 Bad Nauheim, Germany

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Cardiac Surgery Clinic, University of Heidelberg, Im Neuenheimer Feld 110, 69120

Heidelberg, Germany j

Department of Cardiothoracic Surgery, Friedrich Schiller University of Jena, Am Klinikum 1,

07747 Jena, Germany k

Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical

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ACCEPTED MANUSCRIPT School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany l

Department of Cardiac, Thoracic and Vascular Surgery, German Heart Institute,

Augustenburger Platz 1, 13353 Berlin, and DZHK (German Centre for Cardiovascular Research) partner site Berlin, Germany m

Clinic of Cardiac Surgery, Ludwig Maximilian University, Marchioninistrasse 15, 81377

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Munich, Germany n

Zurich Children's Hospital, Department of Congenital Pediatric Surgery, Steinwiesenstrasse

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75, CH 8032 Zurich, Switzerland o

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Clinic for Cardiovascular Surgery, University Hospital Zurich, Rämistrasse 100, 8091

Zurich, Switzerland p

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Department of Cardiac Surgery, Medical University of Vienna, Währinge Gürtel 18-20, A-

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1090 Vienna, Austria

Address for correspondence:

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PD Dr. med. Markus J. Barten

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Universitätsklinikum Hamburg-Eppendorf Martinistraße 52, Gebäude Ost 70

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20246 Hamburg, Germany

Tel: +49 40 7410 -56307 Fax: +49 40 7410 -56417

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Email: [email protected]

Author email addresses:

Markus J. Barten

[email protected]

Uwe Schulz

[email protected]

Andres Beiras-Fernandez

[email protected]

Michael Berchtold-Herz

[email protected]

Udo Boeken

[email protected]

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ACCEPTED MANUSCRIPT [email protected]

Stephan Hirt

[email protected]

Manfred Richter

[email protected]

Arjang Ruhpawar

[email protected]

Tim Sandhaus

[email protected]

Jan Dieter Schmitto

[email protected]

Felix Schönrath

[email protected]

Rene Schramm

[email protected]

Martin Schweiger

[email protected]

Markus Wilhelm

[email protected]

Andreas Zuckermann

[email protected]

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Jens Garbade

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ACCEPTED MANUSCRIPT Conflicts of interest

Markus J. Barten has received speakers honoraria from Therakos as well as honoria as a member of advisory boards for Sanofi, Novartis Pharma and Biotest. Uwe Schulz has received speaker’s honoraria from Sanofi-Genzyme, Novartis and Hexal, travel grants from Biotest and Actelion, and a research grant from Actelion.

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Andres Beiras-Fernandez has received research grants from Sanofi and Orion Pharma and

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has received speakers honoraria from Sanofi Genzyme and Boehringer Ingelheim.

Udo Boeken has no conflicts of interest to declare. Jens Garbade has no conflicts of interest to declare.

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Michael Berchtold-Herz has received honoraria from Sanofi Genzyme.

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Stephan Hirt has received speaker’s honoraria from Novartis.

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Manfred Richter has received speaker’s honoraria from Novartis and Sanofi-Genzyme. Arjang Ruhpawar has no conflicts of interest to declare. Tim Sandhaus has no conflicts of interest to declare.

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Jan Dieter Schmitto has no conflicts of interest to declare.

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Felix Schönrath has received speaker’s honoraria from Abbott, Astra Zeneca, Bayer HealthCare, Novartis and Sanofi-Genzyme.

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Rene Schramm has no conflicts of interest to declare. Martin Schweiger - has received speaker’s honoraria from Novartis and Sanofi-Genzyme.

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Markus J. Wilhelm has received honoraria from Sanofi Genzyme. Andreas Zuckermann has received research grants from Astellas, Roche, Novartis, one Lambda, Chiesi and Sanofi, is a member of the speakers' bureau for Novartis, SanofiGenzyme, Biotest and One Lambda and is a member of advisory boards for Sanofi Genzyme and Sandoz and Biotest.

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ACCEPTED MANUSCRIPT Highlights

Pre-transplant and de novo DSA affect 3–11% and 10–30% of heart transplant patients

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DSA are integral to antibody-mediated rejection, a major cause of graft loss

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DSA are associated with higher graft loss and mortality post-heart transplant

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DSA also appears to play a contributory role in cardiac allograft vasculopathy

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Immunosuppression should minimize de novo DSA risk but data are sparse

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Figure 1

Figure 2

Figure 3

Figure 4