Histological picture of antibody-mediated rejection without donor-specific anti-hla

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1 DR ALEKSANDAR SENEV (Orcid ID : ) MR. MAARTEN COEMANS (Orcid ID : ) Article type : Original Article Histological picture of antibody-mediated rejection without donor-specific anti-hla antibodies: clinical presentation and implications for outcome Authors: Aleksandar Senev 1,2, Maarten Coemans 1, Evelyne Lerut 3, Vicky Van Sandt 2, Liesbeth Daniëls 2, Dirk Kuypers 1,4, Ben Sprangers 1,4, Marie-Paule Emonds 1,2, Maarten Naesens 1,4 *. 1 Department of Microbiology and Immunology, KU Leuven, University of Leuven, Belgium 2 Histocompatibility and Immunogenetics Laboratory, Belgian Red Cross-Flanders, Mechelen, Belgium 3 Department of Pathology, University Hospitals Leuven, Leuven, Belgium 4 Department of Nephrology and Renal Transplantation, University Hospitals Leuven, Leuven, Belgium ORCID Aleksandar Senev, Maarten Coemans, Evelyne Lerut, This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: /ajt.15074

2 Vicky Van Sandt, Liesbeth Daniëls, Dirk Kuypers, Ben Sprangers, Marie-Paule Emonds, Maarten Naesens, Corresponding author: Maarten Naesens, MD, PhD Department of Nephrology and Renal Transplantation, University Hospitals Leuven Herestraat 49, 3000 Leuven, Belgium Tel: ; Fax: Abbreviations ABMR, antibody-mediated rejection; ABMR h, histological picture of ABMR; ah, arteriolar hyalinosis; Bx, biopsy; CS, corticosteroids; C4d, complement split product 4d;

3 cv, vascular intimal thickening; ci, interstitial fibrosis; ct, tubular atrophy; cg, chronic allograft glomerulopathy; DSA, donor-specific antibodies; egfr, estimated glomerular filtration rate; g, glomerulitis; HLA, Human leukocyte antigens; HR, hazard ratio; i, interstitial inflammation; IQR, interquartile range; MFI, median fluorescence intensity; MPA, mycophenolic acid; mvi, microcirculation inflammation; mm, mesangial matrix expansion; ptc, peritubular capillaritis; PTCs, peritubular capillaries; SAB, single antigen bead; t, tubulitis; TAC, tacrolimus;

4 TCMR, T-cell mediated rejection; v, endarteritis ABSTRACT In this cohort study (n=935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody-mediated rejection (ABMR) in the absence of circulating donor-specific anti-hla antibodies (DSA), and compared this to patients with definite ABMR and HLA-DSA-positivity. The histological picture did not differ between HLA-DSA-positive (n=85) and HLA-DSA-negative (n=123) cases of ABMR histology, apart from increased C4d deposition in the peritubular capillaries in HLA-DSA positive cases. Histology of ABMR without HLA-DSA was more transient than DSA-positive ABMR, and patients with ABMR histology without HLA-DSA had superior graft survival than HLA-DSA-positive patients, independent of concomitant TCMR (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR in the absence of HLA-DSA, compared to patients without histological picture of ABMR. Despite an association between C4d deposition and HLA-DSA-positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with histological picture of ABMR but without detectable HLA-DSA represent a distinct, often transient phenotype with superior allograft survival. 1 INTRODUCTION Antibody-mediated rejection (ABMR) was recognized as a distinct diagnostic entity in and is considered as a major cause of late kidney allograft failure. 2,3,4,5 Over the last two decades, significant progress has been made in the diagnosis of ABMR, mainly driven by

5 the development of sensitive assays for the detection of donor-specific antibodies (DSA) against human leukocyte antigens (HLA), and by sequential adjustments of the Banff diagnostic criteria for ABMR. 6 Initially, the three main diagnostic features of ABMR were: (1) morphological evidence of acute tissue injury, (2) presence of DSA and (3) C4d staining in peritubular capillaries (PTCs) as a footprint for complement-mediated graft injury. 1 Later, the discovery that C4d deposition can fluctuate over time led to the recognition of C4d-negative ABMR. 7 In Banff 2015, the three features of ABMR needed to be present for a final ABMR diagnosis. 8 However, in clinical practice, not all cases fulfill these three criteria, and the patients with such an incomplete phenotype are classified as suspicious for ABMR. These cases remain poorly studied, but can essentially be classified into two categories: 1) patients with DSA and some histological lesions of ABMR, but not meeting the full histologic criteria for ABMR, and 2) patients meeting the histological criteria for ABMR but without detectable DSA. The latter category could be explained by presence of injurious antibodies that remain undetected due to the limitation of the current ing methods and to the fact that these methods only focus on circulating anti-hla antibodies, thereby missing non-hla antibodies or by the presence of HLA-specific memory B-cells. 9,10,11,12,13,14,15 Uncertainty about the clinical significance of cases that are suspicious for ABMR has important implications for the management of patients, as it remains unclear whether they require additional treatment for ABMR. The Banff 2017 consensus proposed considering C4d-positivity as alternative for the DSA criterion in cases where DSA ing is not available or potentially false negative. 16 These modifications will expand the number of patients that are diagnosed with ABMR, and eliminate the category suspicious for ABMR. However, it remains unclear whether these patients with the histological picture of ABMR but without

6 circulating HLA-DSA have a different presentation and association with outcome than patients with circulating HLA-DSA. Therefore, in this study we investigated the phenotype and graft outcome of patients who meet the first two Banff 2015 histological criteria for ABMR (ABMR h ) but without circulating HLA-DSA (DSA neg ABMR h ) and of patients with definite ABMR (DSA pos ABMR h ). In addition, we evaluated whether C4d-positivity in PTCs could be used as a proxy for circulating HLA- DSA, as proposed by Banff MATERIALS AND METHODS 2.1 Study population and data collection All consecutive adult recipients of a kidney transplant at the University Hospitals-Leuven between March 2004 and February 2013 were eligible for this study. Recipients of combined transplantations or kidney transplantations after another solid organ transplantation were excluded. All transplantations were performed with negative complement-dependent cytotoxicity cross-matches. The clinical data were prospectively collected during routine clinical follow-up. This study was approved by the Ethics Committee of the University Hospitals-Leuven (S53364). 2.2 Detection of circulating anti-hla antibodies and donor-specificity Pre- and post-transplant anti-hla antibodies were systematically monitored in one histocompatibility laboratory (HILA-Red Cross-Flanders). For the need of this study, routinely bio-banked sera were reed for presence of circulation HLA-DSA in the same laboratory (Table S1). Antibodies against HLA-A,-B,-C,-DRB1,-DRB345,-DQ and -DP loci in the

7 recipient sera were determined for HLA-DSA at the resolution level of single antigen beads (SAB). High-resolution typing of the donor was performed to determine true donor specificity. A positive result for circulation HLA-DSA was defined as a median fluorescence intensity (MFI) of the donor-specific bead above 500, and the highest MFI value of the immunodominant HLA-DSA was reported. We did not for non-hla-dsa. 2.3 Biopsies and histological scoring All post-transplant renal allograft biopsies performed in this cohort, until time of data extraction on November 2017, were included. Protocol-specified biopsies were performed in addition to clinically indicated biopsies. Protocol kidney transplant biopsies were performed at time of transplantation, and at 3, 12 and 24 months after transplantation. In addition, patients who were transplanted before October 2005 were invited for a protocol biopsy performed at 48 months, patients transplanted before November 2008 for a protocol-bx at 36 months and patients transplanted before January 2010 for a protocol-bx at 60 months. An immunohistochemical C4d stain (monoclonal antibody, dilution 1:500, Quidel Corporation, Santa Clara,CA) was performed on frozen tissue. One pathologist (EL) reviewed all biopsies. The severity of the histological lesions was semi-quantitatively scored according to the Banff categories. For complement C4d deposition (C4d) in PTCs, we used the following semi-quantitatively scores: C4d0 no staining (0%), C4d1 minimal staining (>0%<25% of PTCs), C4d2 focal staining (25-75% of PTCs) and C4d3 diffuse staining (>75% of PTCs). Diagnosis of the histological phenotypes was based on the criteria as defined by the Banff 2015 and 2017 consensus. 8,16 We used the term ABMR h for cases that met the first 2 Banff 2015 and 2017 criteria for histology of ABMR by the combination of Banff scores for microvascular inflammation (glomerulitis and peritubular capillaritis), arteritis, thrombotic microangiopathy and C4d deposition, using the first 2 criteria of the Banff classification. 8,16 This definition of ABMR h

8 was made without taking into account the third criterion (serologic evidence of DSA and/or C4d staining in the Banff 2017 update). Cases with ABMR h could be HLA-DSA positive or HLA-DSA negative, C4d positive or C4d negative, and could have concomitant borderline changes or TCMR. The definition of ABMR h was strictly confined to the first 2 criteria of the Banff diagnosis of ABMR (histologic evidence of acute tissue injury plus evidence of current/recent antibody interaction with vascular endothelium), according to Banff 2015 or Banff ,16 Biopsies that did not meet the first 2 criteria of the Banff definition of ABMR, i.e. biopsies that were not classified as ABMR h, were categorized as NoABMR h. Also these cases could have HLA-DSA positive or HLA-DSA negative, C4d positive or C4d negative, and could have concomitant borderline changes or TCMR. Furthermore, borderline changes were defined as foci of tubulitis (t>0) with minor interstitial inflammation (i0 or i1), or moderate-severe interstitial inflammation (i2 or i3) with mild (t1) tubulitis. 8,16 Borderline changes were not included in the TCMR designation, in concordance with the Banff classification. 8, Statistical Analysis When comparing the different groups, the 2-sample t- was used for continuous variables, the Pearson s chi-square for categorical data and Wilcoxon for the comparison of medians. For comparing the histological lesions between groups both t- and chi-square were used. Kidney allograft survival was plotted using the Kaplan-Meier method and groups were compared by log-rank ing. Univariate and multivariate Cox models were applied to quantify the hazard ratios for allograft loss. In case of death with a functioning graft, we censored graft survival at time of death. All s were two-sided and p-values less

9 than 0.05 were considered statistically significant. We used SAS software (version 9.4;SASinstitute). 3 RESULTS 3.1 Study population characteristics Between 2004 and 2013, 1137 patients were transplanted. Patients with combined transplants, with a history of another organ transplantation before the kidney transplantation or with absence of antibody/biopsy follow-up data were excluded, leaving 935 transplant recipients available for this study (Figure 1). The median follow-up time was 6.6 years (IQR: 4.5 years). Baseline and clinical characteristics of the study population are summarized in Table 1. In total, 4295 biopsies were performed and assessed using the Banff classification: 3458 protocol-specified biopsies and 837 indication biopsies. In 208 patients, at least one biopsy met the 2 first Banff 2015 histological criteria for active ABMR (ABMR h ): histological evidence of acute tissue injury (g>0, ptc>0 or v>0) and evidence of current/recent antibody interaction with vascular endothelium (at least one: C4d>0 or [g+ptc 2], with g 1 in cases with acute TCMR, borderline infiltrate or infection). 8 Of these 208 patients, 201 met these criteria by presence of microcirculation inflammation (mvi): 102 had g 1 and ptc 1 together; 99 had g or ptc 1 (63 had g 1 and ptc=0; 36 had g=0 and ptc 1), in addition to other morphologic Banff criteria for ABMR. Only 7 patients met the histological ABMR criteria by positivity for v 1 and C4d>1 in the absence of mvi. Of all 208 patients with ABMR h, only 7 received specific ABMR treatment.

10 3.2 Clinical phenotype of DSA neg ABMR h versus DSA pos ABMR h Anti-HLA antibodies were detected in 104/208 cases with ABMR h. The Banff 2015 criterion of DSA-positivity prior or at the time of the index biopsy (the first biopsy with ABMR h ) was met in 85/208 cases (40.9%; DSA pos ABMR h group). All patients with HLA-DSA with MFI 500 were allocated to the DSA pos ABMR h group, 64 had MFI 2000, while 21 had MFI between (Figure 1). HLA-DSA were present already at time of transplantation in 71/85 patients. De novo-dsa occurred after transplantation in 14 patients prior reaching the histological criteria for ABMR, and two patients with preexisting-dsa additionally developed de novo-dsa with different HLA-specificity. HLA-DSA were absent in 123/208 patients, which means that 59.1% of the cases that met the Banff histological criteria for ABMR did not meet the DSA criterion (DSA neg ABMR h group). Patients with HLA-DSA were more often female, had received a repeat transplantation more frequently, and experienced more delayed graft function than HLA-DSA-negative (Table 1). DSA neg ABMR h had significantly more HLA-A/-B/-DR mismatches, specifically in locus A. 3.3 Histological phenotype of DSA neg ABMR h and DSA pos ABMR h We then compared the histological appearance of the index biopsies of the DSA neg ABMR h and DSA pos ABMR h patients, according to the individual Banff features (Table 2). The main difference in histology between the two groups was C4d deposition in PTCs, which was more prevalent in the DSA pos ABMR h group.

11 The distribution of cases according to C4d-grade, mvi and concomitant TCMR or borderline changes is provided in Tables S2-S3. Thirty-eight percent of cases that met the histological criteria of ABMR in the absence of HLA-DSA had TCMR but these numbers were not different compared to the HLA-DSA-positive patients. Of the DSA neg ABMR h cases, 31.7% did neither have C4d deposition, nor TCMR or borderline changes. Subsequent biopsies after the index-bx indicated that patients with DSA pos ABMR h had significantly more chance of having ABMR h in the next biopsies compared to DSA neg ABMR h patients (Table 2 and Tables S4-S6). The overall number of biopsies that met the histological criteria for ABMR was significantly higher in the DSA pos ABMR h group, and also the number of biopsies with chronic ABMR was higher in the HLA-DSA-positive patients. Given the association between HLA-DSA and C4d in this cohort, we evaluated the diagnostic performance of C4d-positivity for presence of HLA-DSA at different thresholds. 17 In the patients with ABMR h, 47 of 85 patients with HLA-DSA 500 had C4d-positivity (C4d 1), leading to a sensitivity of 55.3% of C4d-positivity for HLA-DSA presence and a positive predictive value of 50.0% (Table 3). 3.4 Graft survival impact of DSA neg ABMR h versus DSA pos ABMR h Figure 2A depicts the Kaplan-Meier survival analysis of the comparison between the DSA neg ABMR h and DSA pos ABMR h groups. Graft survival at 10-years after the index-bx with ABMR h, was significantly better in the DSA neg ABMR h group than in the DSA pos ABMR h group. Adjusted multivariate model-1, confirmed this with a 2.9-fold increased hazard ratio (HR) for allograft failure in the DSA pos ABMR h group compared to the DSA neg ABMR h group (Table 4).

12 Additionally, we evaluated whether C4d-positivity in the DSA neg ABMR h group could be used as proxy for HLA-DSA presence in association with worse graft outcome. Of the DSA neg ABMR h cases, 47 had C4d 1 (DSA neg ABMR h C4d pos ), while 76 patients had C4d=0 (DSA neg ABMR h C4d neg group). The treatment with high dose intravenous corticosteroids was not different (p=0.23) between the C4d-positive (n=25, 53.2%) and C4d-negative (n=32, 42.1%) HLA-DSA-negative cases. Survival analysis illustrated that 10-year graft survival in these cases was not dependent on C4d status or concomitant TCMR. Compared to DSA pos ABMR h patients, significantly higher survival rates were found in both HLA-DSAnegative groups (Figure 2 and Figure S1). Adjusted multivariate model-3 showed a significantly increased risk of graft failure only in DSA pos ABMR h cases (HR=2.9; p=0.007) compared to DSA neg ABMR h C4d neg cases. When the multivariate models were repeated in the subgroup of patients who received their first graft, or with a different threshold for C4d-positivity (C4d>1), DSA neg ABMR h remained associated with better graft outcome (Table 4). 3.5 Graft outcomes in patients with ABMR h versus without, within the first year after transplantation To evaluate the impact of ABMR h on graft outcome, in comparison to NoABMR h patients, we performed survival analyses starting 1-year after transplantation in 897 patients. Thirty-eight patients who lost their graft within the first year after transplantation were excluded from this analysis. The most common causes of graft loss in these patients were death with a functioning graft (15 cases, 39.5%), postoperative complications/infection but without histological reason (12 cases, 31.6%) and in 9 cases the graft lost could be linked to histological reason (3 in DSA pos ABMR h and 6 in NoABMR h group). The distribution of these patients according our grouping was 5 patients in DSA neg ABMR h, 5 in DSA pos ABMR h group and 28 with NoABMR h. Of the 897 patients, 149 patients had at least one biopsy within the first year after transplantation that met the Banff 2015 histological criteria for ABMR. Of

13 these, 66 had HLA-DSA with MFI>500 and were allocated to the DSA pos ABMR h group; 83 patients did not have HLA-DSA and were allocated to the DSA neg ABMR h group. Of these, 35 patients had C4d 1 (DSA neg ABMR h C4d pos ), while 48 had C4d=0 (DSA neg ABMR h C4d neg ). The other 748 patients did not develop ABMR h (NoABMR h group) in the first year, while 116 of them experienced at least one episode of TCMR (NoABMR h TCMR pos ). Kaplan-Meier analysis for outcome after the first-year post-transplant showed that survival in the DSA neg ABMR h group was similar to survival in the NoABMR h group (p=0.73, Figure 3A) and the group with only TCMR (p=0.45, Figure 3B). The outcome of the DSA pos ABMR h group was significantly worse compared to other groups (DSA neg ABMR h, TCMR pos NoABMR h and NoABMR h ) (Figure S2A and S2B). The additional survival analyses confirmed that HLA- DSA-negative cases, independent of the C4d status or concomitant TCMR, had similar outcomes as the NoABMR h patients (Figure 3C and Figure S2C). In univariate analysis (Table 5), female recipient gender and older recipient age, repeat transplantation, delayed graft function, lack of induction therapy, presence of anti-hla antibodies, TCMR, ABMR h and HLA-DSA were significantly associated with graft failure (p<0.05). In the subsequent multivariate models, adjusted for the variables that were significant in univariate analyses (p<0.05), we evaluated the impact of ABMR h and HLA-DSA on the graft survival (Table 6). Only the presence of HLA-DSA was independently associated with an increased risk of graft failure. After regrouping the patients according to the Banff 2015 classification, only patients in the DSA pos ABMR h group had an increased risk of graft failure, and not the patients that were classified as suspicious for ABMR (DSA neg ABMR h ). In this analysis, the risk of graft failure did not differ between the DSA neg ABMR h group and the NoABMR h group. In the final model-

14 4, with patient classification according to the most recent Banff 2017 classification, the DSA neg ABMR h C4d pos and DSA neg ABMR h C4d neg had comparable graft survival and significantly better than the DSA pos ABMR h group (Table 6). Finally, when we evaluated graft functional outcome with the composite endpoint of 50% graft functional decline or graft failure, exactly the same conclusions were obtained in the survival analysis (Figure 4) as well as for the univariate and multivariate models (Tables S7 and S8). 4. DISCUSSION We described the phenotype of kidney transplant biopsies that have the histological picture of ABMR (ABMR h ), in the absence of detectable HLA-DSA. Despite the clear ABMR h, the risk of graft failure was significantly lower in these patients in comparison to patients with similar histology but with HLA-DSA. In the absence of HLA-DSA, the outcome of patients with ABMR h was not worse than in patients without histological suggestion of ABMR. HLA- DSA-negative ABMR h was more transient than HLA-DSA-positive ABMR h, with less development of chronicity. Part of these cases could be related to concomitant TCMR, but the subgroup of patients with ABMR h in the absence of HLA-DSA and TCMR remains poorly understood. We confirmed the association between HLA-DSA and C4d deposition in PTCs but adding the C4d status to the diagnostic classification of ABMR did not determine the prognosis of patients with ABMR h in the absence of HLA-DSA. According to these data, using C4d deposition in PTCs as a proxy for circulating DSA, as suggested in the most recent update of the Banff classification, needs to be interpreted cautiously. 16 Although the Banff consensus has suggested classifying biopsies with ABMR h but without detectable circulating antibodies as suspicious for ABMR since 2005, there is no literature on the phenotype and outcome of this entity. In the current study, we demonstrate that this HLA-DSA-negative ABMR h is observed relatively frequently after transplantation, and that

15 the number of these cases even outnumbered the number of cases with detectable HLA- DSA. Interestingly, apart from significantly less C4d deposition in PTCs, patients with ABMR h in the absence of HLA-DSA also had a different evolution after their index biopsy. These patients had significantly less subsequent biopsies with ABMR h than HLA-DSApositive patients, and less development of chronicity. It is well described that ABMR due to DSA is a waxing and waning process, which ultimately culminates in irreversible chronic lesions and graft failure. 18 Our multivariate models showed that the presence of HLA-DSA is more important for predicting outcome than reaching ABMR h, which corresponds to previous studies that showed an independent association of HLA-DSA and ABMR h with outcome. 19,20,21 This is not the case for HLA-DSA-negative patients with ABMR h, where our study suggests that this histology, in the absence of HLA-DSA, is often a transient phenomenon, without an increased risk of graft functional decline or graft failure. The pathophysiology of these cases with ABMR h without HLA-DSA remains unclear. Of these patients, 38.2% had concomitant TCMR, and it is possible that the mvi we noted is a consequence of T-cell activation and infiltration in the graft, rather than a primary process targeting the graft endothelium. Nevertheless, 31.7% of cases with ABMR h in the absence of HLA-DSA or C4d deposition did not have concomitant TCMR or borderline changes, which makes it unlikely that this phenomenon is the sole explanation for this phenotype. The cooccurrence of ABMR h and TCMR lesions, in mixed types of rejection, could represent different etiologies depending on the HLA-DSA status, and further study of the temporal associations between these phenomena seems relevant to disentangle these complex phenotypes. Further studies are necessary to elucidate this phenotype of HLA-DSA-negative cases of ABMR h, in the absence of TCMR or borderline changes.

16 In addition, also methodological issues associated with DSA detection could explain some cases with ABMR h. We have carefully re-evaluated all anti-hla antibodies using the same sensitive in all patients with ABMR h and have re-typed the donors where necessary. Nevertheless, it is well reported that non-hla-dsa can exist after transplantation and HLAspecific memory B-cells could be found in absence of circulating HLA-DSA. 12,13,10,11 addition, deleterious antibodies may be detected in the graft as was shown by others. 22,23 These phenomena could influence our results and the assessment of non-hla-dsa, of intragraft antibody profiles or of HLA-specific memory B-cells could have explained some of DSA neg ABMR h cases. But the absence of association between HLA-DSA-negative ABMR h with graft outcome suggests that the pathogenicity of such antibodies and cells, and of the potentially associated histological picture, is significantly less than the pathogenicity of detectable circulating HLA-DSA and the associated histological lesions. Hence, it is questionable whether these cases, if diagnosed as ABMR by other criteria than the presence of circulating HLA-DSA, would benefit from additional therapies targeting ABMR. There was no significant difference in graft survival between C4d-negative and C4d-positive patients in DSA neg ABMR h group. In the absence of HLA-DSA, C4d deposition was not associated with worse graft outcome. This finding questions the last Banff 2017 update of the diagnostic criteria for ABMR. 16 There it was proposed to consider C4d-positivity as an alternative for the serologic criterion for DSA neg ABMR h cases. Although studies based on smaller and earlier cohorts (before the era of SAB-ing) supported the conclusion that C4d-positivity is a marker of circulating DSA, 24,25,26 these studies had not evaluated the specificity or positive predictive value of C4d for HLA-DSA presence in cases with ABMR h. To our knowledge, our study is the first to directly evaluate the diagnostic performance of C4d-positivity in this setting with sensitive Luminex-based assays for detection and identification of anti-hla antibodies. Although the DeKAF study illustrated that there was a clear discordance between the presence of C4d and circulating HLA-DSA, formal diagnostic In

17 performance measures were not provided in this study. 27 The fact that the majority of patients in the DeKAF study received specific ABMR therapy, versus virtually none in our study, could also be relevant. Finally, the thresholds for HLA-DSA-positivity could also play a role. However, if we put the threshold of HLA-DSA-positivity at MFI 2000, our data show that the association with graft outcome is even more significant than when we use a threshold of MFI 500. The independent importance of HLA-DSA-positivity for graft outcome is very evident in our study, and using higher thresholds only improves its prognostic relevance. Our conclusion that circulating HLA-DSA are a significant and independent determinant of graft outcome corresponds to other studies that identified circulating HLA-DSA as a major predictor for graft failure, regardless of C4d status, 28 and to studies that found no independent association of C4d staining with graft survival. 29,30 Nevertheless, our finding that the presence of C4d did not contribute to the prognosis of patients with ABMR h is in sharp contrast to the findings in the DeKAF study, where the outcome of patients with new-onset late graft dysfunction was primarily determined by C4d status and not by HLA-DSA status. 27 This discrepancy could be due to the very different study design: we evaluated phenomena early after transplantation in an unselected cohort, whereas the DeKAF study evaluated a selected population of cases with deterioration of graft function late after transplantation. Finally, due to the reported limited reproducibility of C4d-staining and interpretation between centers, 31 increasing the impact of C4d-positivity on the diagnostic criteria for ABMR, as done in the last Banff update, 16 could further decrease reproducibility of the Banff diagnostic scheme between institutions.

18 Our study also has several limitations. For the diagnosis of ABMR h we relied completely on the diagnostic criteria of the Banff classification. Updates of the diagnostic criteria for ABMR h could impact the interpretation of our data. Furthermore, we used the indexbx to compare the lesions and association with graft failure, rather than including all available follow-up biopsy data in this cohort. Further work is needed to include longitudinal aspects and timedependencies in the analyses, which are especially relevant in chronic disease processes like ABMR. We also lack information on non-hla-dsa. The majority of DSA pos ABMR h cases had preexisting-dsa. Recent data suggested that de novo-dsa associate with worse outcome compared to pre-existing DSA, which was confirmed in our study. 32 The overall applicability of our study results beyond patients with preexisting-dsa is not clear, as we primarily included patients with preexisting low-level-dsa. Our inferences on the comparison between ABMR h negative and ABMR h positive groups are valid for patients who develop ABMR h in their first year after transplantation and live until at least the end of the first year. The virtual absence of use of targeted therapies against ABMR obviates drawing conclusions on the potential relevance of therapy in this setting. This deviation from the clinical practice of most transplant centers with regards to treatment is partly explained by the lack of reimbursement for therapies for ABMR in our country. While this could impact the generalizability of our data, the lack of specific therapy and absence of difference in therapeutic approach between HLA-DSA-positive and negative cases, has the advantage that our study represents the natural disease evolution and reinforces the conclusions about the outcome differences which cannot be explained by treatment effects. In summary, patients with ABMR h but without HLA-DSA represent a distinct, often transient phenotype with superior graft survival compared to patients with circulating HLA-DSA and fully developed ABMR by Banff criteria. Although the association between C4d deposition in PTCs and HLA-DSA-positivity is confirmed, using C4d deposition in PTCs as proxy for circulating HLA-DSA, as suggested in the recent update of the Banff classification, did not

19 contribute to the prognosis of graft function and failure, independent of the threshold used. Further work is needed in order to elucidate the pathophysiology of ABMR h in the absence of HLA-DSA. ACKNOWLEDGMENTS AS, MN, MC and MPE are funded by the Research Foundation - Flanders (FWO) and the Flanders Innovation & Entrepreneurship agency (VLAIO), with a TBM project (grant n IWT ; "TEMPLATE"). DISCLOSURE The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. FIGURE LEGEND Figure 1. Patient enrollment and group definition, according to ABMR h, HLA-DSA status and presence of C4d. ABMR h, histological picture of ABMR; HLA, human leukocyte antigen; DSA, donor-specific anti-hla antibodies. Figure 2. Kaplan-Meier survival analysis in the patients with ABMR h diagnosed on at least one post-transplant biopsy (N=208).

20 (A) Survival analysis in the patients with ABMR h grouped according to HLA-DSA status. The 10-year allograft survival rates are: 76.2% for DSA neg ABMR h group and 56.8% for DSA pos ABMR h group (log-rank, p=0.001). (B) Survival analysis in the patients with ABMR h grouped according to HLA-DSA status and C4d status. The 10-year allograft survival rates are: 54.3% for DSA pos ABMR h C4d pos versus 58.0% for DSA pos ABMR h C4d neg (the log-rank is not significant, p=0.49), and 72.5% for DSA neg ABMR h C4d neg versus 78.9% for DSA neg ABMR h C4d pos group (p=0.91). (C) Survival analysis in the patients with ABMR h grouped according to HLA-DSA status and concomitant TCMR. Ten-year survival rates are: 44.0% for DSA pos ABMR h TCMR pos versus 64.8% for DSA pos ABMR h TCMR neg (log-rank, p=0.16), and 78.6% for DSA neg ABMR h TCMR pos versus 71.5% for DSA neg ABMR h TCMR neg group (log-rank, p=0.92). Figure 3. Kaplan-Meier survival analysis for graft survival after the first year posttransplant (N=897). (A) Survival analysis in the patients grouped according to ABMR h and HLA-DSA within the first year post-transplant. The 10-year graft survival was significantly different in the DSA pos ABMR h (57.5%) group, compared to the NoABMR h group (85.0%, log-rank, p<0.0001) and the DSA neg ABMR h group (83.5%, log-rank, p=0.0005). The difference between the DSA neg ABMR h and NoABMR h group was not significant (log-rank, p=0.73). (B) Survival analysis in the patients grouped according to ABMR h, DSA status and TCMR within the first year post-transplant. The 10-year graft survival of NoABMR h TCMR pos (74.9%) and NoABMR h TCMR neg (87.5%) groups reached statistical

21 significance (p=0.05). However, both groups had similar survival as DSA neg ABMR h group (log-rank, p=0.45 and p=0.49, respectively). (C) Survival analysis in the patients grouped according to ABMR h, HLA-DSA status, C4d status and TCMR within the first year post-transplant. Both HLA-DSA-positive groups had similar 10-year graft survival (log-rank, p=0.68), 59.8% for DSA pos ABMR h C4d pos and 48.3% for DSA pos ABMR h C4d neg, but significantly worse compared to all other groups (log-rank s with p<0.05). Figure 4. Kaplan-Meier survival analysis for graft survival with the composite endpoint of graft functional decline by 50% or graft failure after the first year post-transplant (N=874). 23 patients were excluded from this analysis due to no egfr measurements one year after transplantation (1 in DSA pos ABMR h, 2 in DSA neg ABMR h and 20 in NoABMR h group). (A) Survival analysis in the patients grouped according to ABMR h and HLA-DSA status within the first year post-transplant. The 10-year graft survival was significantly different in the DSA pos ABMR h (38.0%) group, compared to the NoABMR h group (73.7%, logrank, p<0.0001) and the DSA neg ABMR h group (70.4%, log-rank, p=0.0001). The difference between the DSA neg ABMR h and NoABMR h group was not significant (log-rank, p=0.71). (B) Survival analysis in the patients grouped according to ABMR h, HLA-DSA status and C4d status within the first year post-transplant. The 10-year allograft survival rates are: 38.8% for DSA pos ABMR h C4d pos versus 32.3% for DSA pos ABMR h C4d neg (p=0.92), and 61.7% for DSA neg ABMR h C4d pos versus 77.7% for DSA neg ABMR h C4d neg group (p=0.20). There was no difference in graft survival between the last two groups and NoABMR h group (logrank, p=0.19 and p=0.53, respectively).

22 SUPPORTING INFORMATION Additional Supporting Information may be found online in the supporting information tab for this article. REFERENCES 1. Racusen LC, Colvin RB, Solez K, et al. Antibody-Mediated Rejection Criteria an Addition to the Banff 97 Classification of Renal Allograft Rejection. American Journal of Transplantation. 2003;3: El-Zoghby ZM, Stegall MD, Lager DJ, et al. Identifying specific causes of kidney allograft loss. American Journal of Transplantation. 2009;9(3): Naesens M, Kuypers DRJ, De Vusser K, et al. The histology of kidney transplant failure: A long-term follow-up study. Transplantation. 2014;98(4): Orandi BJ, Chow EHK, Hsu A, et al. Quantifying Renal Allograft Loss Following Early Antibody-Mediated Rejection. American Journal of Transplantation. 2015;15(2): Sellarés J, Reeve J, Loupy A, et al. Molecular diagnosis of antibody-mediated rejection in human kidney transplants. American Journal of Transplantation. 2013;13(4): De Serres SA, Noël R, Côté I, et al Banff Criteria for Chronic Active Antibody- Mediated Rejection: Assessment in a Real-Life Setting. American Journal of Transplantation. 2016;16(5): Haas M, Sis B, Racusen LC, et al. Banff 2013 meeting report: Inclusion of C4dnegative antibody-mediated rejection and antibody-associated arterial lesions. American Journal of Transplantation. 2014;14(2): Loupy A, Haas M, Solez K, et al. The Banff 2015 Kidney Meeting Report: Current

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25 capillaries. Journal of the American Society of Nephrology. 1999;10(11): Mauiyyedi S, Crespo M, Collins a B, et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. Journal of the American Society of Nephrology : JASN. 2002;13(3): Bohmig G a, Exner M, Habicht A, et al. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol. 2002;13(4): Gaston RS, Cecka JM, Kasiske BL, et al. Evidence for antibody-mediated injury as a Major determinant of late kidney allograft failure. Transplantation. 2010;90(1): Loupy A, Hill GS, Suberbielle C, et al. Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). American Journal of Transplantation. 2011;11(1): Botermans JM, de Kort H, Eikmans M, et al. C4d staining in renal allograft biopsies with early acute rejection and subsequent clinical outcome. Clinical Journal of the American Society of Nephrology. 2011;6(5): Sis B, Jhangri GS, Riopel J, et al. A new diagnostic algorithm for antibody-mediated microcirculation inflammation in kidney transplants. American Journal of Transplantation Mengel M, Chan S, Climenhaga J, et al. Banff initiative for quality assurance in transplantation (BIFQUIT): Reproducibility of C4d immunohistochemistry in kidney allografts. American Journal of Transplantation. 2013;13(5): Aubert O, Loupy A, Hidalgo L, et al. Antibody-Mediated Rejection Due to Preexisting versus De Novo Donor-Specific Antibodies in Kidney Allograft Recipients. Journal of the American Society of Nephrology

26 Table 1. Main demographic and clinical characteristics of the study population. Characteristics Study cohort (n=935) ABMR h (n=208) Recipients DSA pos AB MR h (n=85) DSA neg AB MR h (n=123) p- value Testa Age (years), mean ±SD 53.5 ± ± ± ± t- Gender (female), n (%) Caucasian ethnicity, n (%) Repeat transplantation, n (%) Diabetes mellitus (DM), n (%) Pre-transplant DM, n (%) De novo post-transplant DM, n (%) 363 (38.8%) 92 (40.2%) 45 (52.9%) 47 (38.2%) (98.8%) (98.3%) (97.6%) (96.8%) (26.9%) 44 (51.8%) 12 (9.76%) <0.000 (15.1%) (43.5%) (53.8%) 45 (52.9%) 67 (54.5%) (15.6%) 44 (21.2%) 18 (21.2%) 26 (21.1%) (28.1%) 68 (32.7%) 27 (31.8%) 41 (33.3%) 0.81 Donors Age (years), mean ±SD 47.6 ± ± ± ± t- Gender (female), n (%) Deceased donor, n (%) Donation after brain death, n (%) Cold ischemia time (hours), mean ±SD HLA-A/B/DR mismatches, mean ±SD 437 (46.7%) 878 (93.9%) 729 (78.0%) 93 (32.1%) 45 (52.9%) 47 (38.2%) (92.8%) 162 (77.9%) 79 (92.9%) 114 (92.7%) (80.0%) 94 (76.4%) ± ± ± ± t- Immunological profile at the index biopsy 2.73 ± ± ± ± t- HLA-A mismatches, mean ±SD 0.96 ± ± ± ± t- HLA-B mismatches, mean ±SD 1.04 ± ± ± ± t- HLA-DR mismatches, mean ±SD HLA antibodies, n (%) Donor-specific HLA antibodies, n (%) Delayed graft function, n (%) Immunosuppression regimen: TAC-MPA-CS, n (%) 0.73 ± ± ± ± t- 287 (22.8%) 146 (15.6%) 104 (50%) 85 (100%) 19 (15.5%) < (40.9%) 85 (100%) 0 (0%) 1.00 Treatment at the time of transplantation and follow-up 167 (17.9%) 673 (71.8%) 51 (24.5%) 27 (31.8%) 24 (19.5%) (89.9%) 83 (97.7%) 104 (84.6%) 0.002

27 Induction therapy, n (%) Proteinuria at time of ABMR h (g/24h), mean ±SD / 0.45 ± ± ± t- egfr at time of ABMR h (ml/min/1.73m 2 ), mean ±SD / 34.9 ± ± ± t- a The 2-sample t- was used for continuous variables, the Pearson s chi-square for categorical variables and the Wilcoxon for the comparison of medians. DSA, donor specific antibodies; ABMR, antibody-mediated rejection; TAC, tacrolimus; MPA, mycophenolic acid; CS, corticosteroids; SD, standard deviation; IQR, interquartile range; IV, intravenous; ATG, anti-thymocyte globulins; ABMR h, histological picture of ABMR; egfr: estimated glomerular filtration rate; HLA, human leukocyte antigens; MFI, median fluorescence intensity. 324 (34.5%) 107 (51.4%) 61 (71.8%) 46 (37.4%) < Graft failure, n (%) (22.6%) 28 (32.9%) 19 (15.4%) Overall treatment after the index biopsy, n (%) High dose IV corticosteroids, n (%) Treatment after the index biopsy / / 113 (54.3%) 101 (48.6%) 51 (60.0%) 62 (50.4%) (51.8%) 57 (46.3%) 0.44 Treatment with ATG, n (%) / 9 (4.3%) 6 (7.1%) 3 (2.4%) 0.11 Specific ABMR therapy, n (%) / 7 (3.4%) 4 (4.7%) 3 (2.4%) 0.37 Overall treatment after the index biopsy (indication biopsies only), n (%) High dose IV corticosteroids, n (%) / 95 (83.3%) 42 (87.5%) 53 (80.3%) 0.31 / 87 (76.3%) 36 (75.0%) 51 (77.3%) 0.78 Treatment with ATG, n (%) / 8 (7.0%) 6 (12.5%) 2 (3.0%) 0.07 Specific ABMR therapy, n (%) / 6 (6.1%) 4 (8.3%) 2 (3.0%) 0.22 Median time to index biopsy (days), (IQR) Diagnosis at indication biopsy, n (%) Graft characteristics at time of ABMR h / 78 (358) 40 (347) 88 (381) 0.20 / 114 (54.8%) Wilco xon 48 (56.5%) 66 (53.7%) 0.69 Diagnosis at protocol biopsy, n (%) / 94 (45.2%) 37 (43.5%) 57 (46.3%) 0.69

28 Table 2. Comparison of the histological appearance of the ABMR h on the index biopsies, according to HLA-DSA status. Histology Individual histological lesions DSA pos ABM R h (n=85) DSA neg ABM R h (n=123) p-value Test a g score, mean ±SD 1.6 ± ± t- g 1, n(%) 65 (76.5%) 100 (81.3%) 0.40 ptc score, mean ±SD 1.15 ± ± t- ptc 1, n(%) 65 (76.5%) 84 (68.3%) 0.20 mvi score, mean ±SD 2.75 ± ± t- mvi 2, n (%) 75 (88.2%) 107 (86.9%) 0.79 C4d score, mean ±SD 1.4 ± ± t- C4d 1, n (%) 47 (55.3%) 47 (38.2%) 0.02 C4d 2, n (%) 40 (47.1%) 29 (23.6%) v score, mean ±SD 0.39 ± ± t- v 1, n(%) 32 (37.7%) 41 (33.3%) 0.52 cg score, mean ±SD 0.06 ± ± t- cg 1, n(%) 3 (3.6%) 6 (4.9%) 0.64 i score, mean ±SD 1.06 ± ± t- i 1, n(%) 40 (47.1%) 62 (50.4%) 0.63 t score, mean ±SD 0.86 ± ± t- t 1, n(%) 51 (60.0%) 77 (62.6%) 0.70 ci score, mean ±SD 0.41 ± ± t- ci 1, n(%) 23 (27.1%) 35 (28.5%) 0.83 ct score, mean ±SD 0.59 ± ± t- ct 1, n(%) 44 (51.76%) 82 (66.7%) 0.03 mm score, mean ±SD 0.09 ± ± t- mm 1, n(%) 7 (8.2%) 10 (8.1%) 0.98 ah score, mean ±SD 0.39 ± ± t- ah 1, n(%) 24 (28.2%) 39 (31.7%) 0.59 cv score, mean ±SD 0.72 ± ± t- cv 1, n(%) 45 (52.9%) 55 (44.72%) 0.24

29 Overall histology Concomitant borderline changes, n (%) 7 (8.2%) 22 (17.9%) 0.05 Concomitant T-cell mediated rejection, n (%) 35 (41.2%) 47 (38.2%) 0.67 Grade I, n (%) 7 (8.2%) 10 (8.1%) 0.98 Grade II, n (%) 28 (32.9%) 37 (30.1%) 0.67 Overall number of biopsies, mean ±SD 5.04 ± ± t- Number of patients with follow-up 79 (92.9%) 109 (88.6%) 0.30 biopsies Overall number of biopsies after the 2.67 ± ± t- index biopsy, mean ±SD Overall number of biopsies with ABMR h, 2.27 ± ± t- mean ±SD Patients with ABMR h at the next biopsy after the index biopsy, n (%) b 41 (51.9%) 29 (26.6%) Patients with cg at the next biopsy after the index biopsy, n (%) b 9 (11.4%) 4 (3.7%) 0.04 Patients with chronic ABMR histology development after the index biopsy, n 13 (16.5%) 9 (8.3%) 0.08 (%) b Patients with cg development after the index biopsy, n (%) b 21 (26.6%) 18 (16.5%) 0.09 Patients with de novo DSA occurrence 5 (5.9%) 6 (4.9%) 0.75 after the index biopsy, n (%) a The 2-sample t- was used for continuous variables, the Pearson s chi-square for categorical variables. b Follow-up biopsies after the index biopsy were not available in all cases: 20 recipients were without next biopsy (6 DSA pos and 14 DSA neg ), and 4 recipients experienced graft loss within the next 60 days (1 DSA pos and 3 DSA neg ). g, glomerulitis; ptc, peritubular capillaritis; v, endarteritis; i, interstitial inflammation; t, tubulitis; cg, chronic allograft glomerulopathy; C4d, C4d complement fraction deposition in peritubular capillaries; mvi, microcirculation inflammation; ci, interstitial fibrosis; ct, tubular atrophy; mm, mesangial matrix expansion; ah, arteriolar hyalinosis; cv, vascular intimal thickening, ABMR h, histological picture of ABMR.

30 Table 3. Association with and diagnostic performance of C4d deposition for HLA-DSApositivity in the ABMR h index biopsies (N=208). P values were calculated using the Chi square. Parameter HLA-DSA MFI threshold 500 HLA-DSA MFI threshold 2000 C4d deposition neg pos neg pos neg pos neg pos C4d score 0 1, 2, 3 0, 1 2, 3 0 1, 2, 3 0, 1 2, 3 DSA threshold 38 (33.3%) 47 (50.0%) 45 (32.4%) 40 (58.0%) 28 (24.6%) 36 (38.3%) 34 (24.5%) 30 (43.5%) DSA < threshold 76 (66.7%) 47 (50.0%) 94 (67.6%) 29 (42.0%) 86 (75.4%) 58 (61.7%) 105 (75.5%) 39 (56.5%) p-value Odds ratio ( ) ( ) ( ) ( ) Accuracy (%) 59.1% 64.4% 58.7% 64.9% Sensitivity (%) 55.3% 47.1% 56.3% 46.9% Specificity (%) 61.8% 76.4% 59.7% 72.5% Positive predictive value (%) Negative predictive value (%) 50.0% 58.0% 38.3% 43.5% 66.8% 67.6% 75.4% 75.5% Neg, negative; pos, positive; DSA, donor specific antibodies; C4d, C4d complement fraction deposition in peritubular capillaries; MFI, median fluorescence intensity.

31 Table 4. Univariate and multivariate Cox proportional hazards analyses for graft failure after the index biopsy in the patients with ABMR h, censored for recipient death (N=208). Variables No. of patients No. of events HR 95% CI p-value Univariate analysis Recipient with diabetes mellitus No Yes Donor age Recipient age Repeat transplantation No Yes Delayed graft function No Yes Cold ischemia time (hours) Immunosuppression: TAC-MPA-CS No Yes Induction therapy at transplantation No Yes Rejection treatment after the index biopsy No Yes Time till index biopsy (days) Protocol or indication biopsy Protocol biopsy Indication biopsy Concomitant TCMR No Yes Concomitant borderline changes No Yes C4d positivity (C4d>0) Negative (C4d0) Positive (C4d1, C4d2 & C4d3) C4d positivity (C4d>1) Negative (C4d0 & C4d1) Positive (C4d2 & C4d3) Timing of HLA-DSA till the index biopsy No DSA Preexisting DSA De novo DSA ABMR (Banff 2015 criteria) Absent DSA neg ABMR h Present DSA pos ABMR h

32 ABMR (Banff 2017 criteria, C4d>0) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h ) ABMR (Banff 2017 criteria, C4d>1) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h ABMR only in 1 st graft recipients (Banff 2015 criteria) Absent DSA neg ABMR h Present DSA pos ABMR h ABMR only in 1 st graft recipients (Banff 2017 criteria, C4d>0) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h ABMR only in 1 st graft recipients (Banff 2017 criteria, C4d>1) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h Multivariate model-1 ABMR (Banff 2015 criteria) Absent DSA neg ABMR h Present DSA pos ABMR h Multivariate model-2 ABMR (Banff 2015 criteria) Absent DSA neg ABMR h Present DSA pos ABMR h (preexisting-dsa) Present DSA pos ABMR h (de novo-dsa) Multivariate model-3 ABMR (Banff 2017 criteria, C4d>0) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h Multivariate model-4 ABMR (Banff 2017 criteria, C4d>1) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h Multivariate model-5 (only 1 st graft recipients) ABMR (Banff 2015 criteria) Absent DSA neg ABMR h Present DSA pos ABMR h Multivariate model-6 (only 1 st graft recipients) ABMR (Banff 2017 criteria, C4d>0) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h Multivariate model-7 (only 1 st graft recipients)

33 ABMR (Banff 2017 criteria, C4d>1) Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h All multivariate Cox models were adjusted for time after transplantation until the index biopsy, concomitant TCMR, repeat transplantation and recipient age. HR, hazard ratio; CI, confidence interval; HLA, human leukocyte antigens; DSA, donorspecific antibodies; ABMR, antibody-mediated rejection; ABMR h, histological picture of ABMR.

34 Table 5. Univariate Cox proportional hazards analyses for graft failure after the first year post-transplant, censored for recipient death (N=897). Variables No. of No. of patients events HR 95% CI p-value Recipient gender Male Female Rec age Repeat transplantation No Yes Recipient with diabetes mellitus No Yes Donor gender Male Female Donor age Type of donor Living donor Diseased donor Type of deceased donor Brain dead Cardiac dead Delayed graft function No Yes Cold ischemia time (hours) Immunosuppression: TAC-MPA-CS Yes No Induction therapy No Yes anti-hla antibodies No Yes < HLA-DSA (MFI>500 until 1 st year) No Yes < HLA-DSA (MFI until 1 st year) MFI < MFI > 500 < MFI <.0001 Timing of HLA-DSA (until 1 st year) No DSA Preexisting-DSA <.0001 De novo-dsa ABMR h (ABMR h until 1 st year)

35 No Yes < TCMR (until 1 st year) No Yes Borderline changes (until 1 st year) No Yes ABMR (Banff 2015 criteria) Absent NoABMR h Absent DSA neg ABMR h Present DSA pos ABMR h < ABMR (Banff 2017 criteria) Absent NoABMR h Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h < HR, hazard ratio; CI, confidence interval; TAC: tacrolimus; MPA: mycophenolic acid; CS: corticosteroids; HLA, human leukocyte antigens; DSA, donor-specific antibodies; ABMR, antibody-mediated rejection; ABMR h, histological picture of ABMR; TCMR, T cell mediated rejection.

36 Table 6. Multivariate Cox proportional hazards models for graft failure after the first year post-transplant according to DSA status, ABMR h and presence of C4d in peritubular capillaries, censored for recipient death (N=897). Variables No. of patients No. of events HR 95% CI P value Multivariate model-1 HLA-DSA (MFI until 1 st year) MFI < MFI >500 < MFI ABMR h (ABMR h until 1 st year) No Yes Multivariate model-2 Timing of HLA-DSA (until 1 st year) No DSA Preexisting-DSA De novo-dsa ABMR h (until 1 st year) No Yes Multivariate model-3 ABMR (Banff 2015 criteria) Absent NoABMR h Absent DSA neg ABMR h Present DSA pos ABMR h Multivariate model-4 ABMR (Banff 2017 criteria) Absent NoABMR h Absent DSA neg ABMR h C4d neg Present DSA neg ABMR h C4d pos Present DSA pos ABMR h All models were adjusted for the variables significant in univariate analysis (p<0.05): recipient gender, recipient age, repeat transplantation, delayed graft function and induction therapy and concomitant TCMR. HR, hazard ratio; CI, confidence interval; HLA, human leukocyte antigens; DSA, donorspecific HLA antibodies; ABMR, antibody-mediated rejection; ABMR h, histological picture of ABMR; TCMR, T-cell mediated rejection.

37 Figure 1. Patient enrollment and group definition, according to ABMR h, HLA-DSA status and presence of C4d.