Risk factors, histopathological features, and graft outcome of transplant glomerulopathy in the absence of donor-specific HLA antibodies
Authors: Aleksandar Senev, Elisabet Van Loon, Evelyne Lerut, Jasper Callemeyn, Maarten Coemans, Vicky Van Sandt, Dirk Kuypers, Marie-Paule Emonds, Maarten Naesens
Journal: Kidney International (2021)

Transplant glomerulopathy is established as a hallmark of chronic antibody-mediated rejection in kidney transplant patients with donor-specific HLA antibodies (HLA-DSA). The clinical importance of transplant glomerulopathy in the absence of HLA-DSA is not well established. To help define this, 954 patients (encompassing 3744 biopsies) who underwent kidney transplantation 2004-2013 were studied with retrospective high-resolution HLA genotyping of both donors and recipients. The risk factors, histopathological appearance and prognosis of cases with transplant glomerulopathy in the absence of HLA-DSA were compared to those cases with HLA-DSA, and the impact of the PIRCHE-II score and eplet mismatches on development of transplant glomerulopathy evaluated. In this cohort, 10.3% developed transplant glomerulopathy, on average 3.2 years post-transplant. At the time of glomerulopathy, 23.5% had persistent pre-transplant or de novo HLA-DSA, while 76.5% were HLA-DSA negative. Only HLA-DSA was identified as a risk factor for glomerulopathy development as eplet mismatches and the PIRCHE-II score did not associate. HLA-DSA negative biopsies with glomerulopathy had less interstitial inflammation, less glomerulitis, and less C4d deposition in the peritubular capillaries compared to the HLA-DSA positive biopsies with glomerulopathy. While graft function was comparable between the two groups, HLA-DSA positive glomerulopathy was associated with a significantly higher risk of graft failure compared to HLA-DSA negative glomerulopathy (Hazard Ratio 3.84; 95% confidence interval 1.94-7.59). Landmark analysis three-years post-transplant showed that HLA-DSA negative patients with glomerulopathy still had a significant increased risk of graft failure compared to patients negative for glomerulopathy (2.62; 1.46-4.72). Thus, transplant glomerulopathy often occurs in the absence of HLA-DSA, independent of HLA molecular mismatches, and represents a different phenotype with less concomitant inflammation and better graft survival compared to that developed in the presence of HLA-DSA. Transplant glomerulopathy is established as a hallmark of chronic antibody-mediated rejection in kidney transplant patients with donor-specific HLA antibodies (HLA-DSA). The clinical importance of transplant glomerulopathy in the absence of HLA-DSA is not well established. To help define this, 954 patients (encompassing 3744 biopsies) who underwent kidney transplantation 2004-2013 were studied with retrospective high-resolution HLA genotyping of both donors and recipients. The risk factors, histopathological appearance and prognosis of cases with transplant glomerulopathy in the absence of HLA-DSA were compared to those cases with HLA-DSA, and the impact of the PIRCHE-II score and eplet mismatches on development of transplant glomerulopathy evaluated. In this cohort, 10.3% developed transplant glomerulopathy, on average 3.2 years post-transplant. At the time of glomerulopathy, 23.5% had persistent pre-transplant or de novo HLA-DSA, while 76.5% were HLA-DSA negative. Only HLA-DSA was identified as a risk factor for glomerulopathy development as eplet mismatches and the PIRCHE-II score did not associate. HLA-DSA negative biopsies with glomerulopathy had less interstitial inflammation, less glomerulitis, and less C4d deposition in the peritubular capillaries compared to the HLA-DSA positive biopsies with glomerulopathy. While graft function was comparable between the two groups, HLA-DSA positive glomerulopathy was associated with a significantly higher risk of graft failure compared to HLA-DSA negative glomerulopathy (Hazard Ratio 3.84; 95% confidence interval 1.94-7.59). Landmark analysis three-years post-transplant showed that HLA-DSA negative patients with glomerulopathy still had a significant increased risk of graft failure compared to patients negative for glomerulopathy (2.62; 1.46-4.72). Thus, transplant glomerulopathy often occurs in the absence of HLA-DSA, independent of HLA molecular mismatches, and represents a different phenotype with less concomitant inflammation and better graft survival compared to that developed in the presence of HLA-DSA. Transplant glomerulopathy (TG), duplication or multilayering of the glomerular basement membrane, is considered to represent the morphologic reflection of chronic or repetitive injury to the glomerular endothelium of renal allografts.1Van Loon E. Lerut E. Naesens M. The time dependency of renal allograft histology.Transpl Int. 2017; 30: 1081-1091Crossref PubMed Scopus (11) Google Scholar,2Filippone E.J. McCue P.A. Farber J.L. Transplant glomerulopathy.Mod Pathol. 2018; 31: 235-252Crossref PubMed Scopus (19) Google Scholar While its pathogenesis is essentially unclear, and the clinical presentation is heterogeneous, the diagnosis of TG is associated with kidney graft functional deterioration and shortened allograft survival.3Naesens M. Kuypers D.R.J. De Vusser K. et al.Chronic histological damage in early indication biopsies is an independent risk factor for late renal allograft failure.Am J Transplant. 2013; 13: 86-99Crossref PubMed Scopus (46) Google Scholar, 4Baid-Agrawal S. Farris A.B. Pascual M. et al.Overlapping pathways to transplant glomerulopathy: chronic humoral rejection, hepatitis C infection, and thrombotic microangiopathy.Kidney Int. 2011; 80: 879-885Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5Naesens M. Lerut E. Emonds M. et al.Proteinuria as a noninvasive marker for renal allograft histology and failure: an observational cohort study.J Am Soc Nephrol. 2016; 27: 281-292Crossref PubMed Scopus (42) Google Scholar To date, no effective therapeutic approaches have been able to reverse the poor outcomes of kidney transplants with TG.6Patri P. Seshan S.V. Matignon M. et al.Development and validation of a prognostic index for allograft outcome in kidney recipients with transplant glomerulopathy.Kidney Int. 2016; 89: 450-458Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Therefore, there is an unmet need to better understand the causes and effector mechanisms of the different phenotypes of TG that can lead to the development of new therapeutics for TG and subsequently to improved kidney graft outcome. Improvements in HLA antibody detection methods have allowed the investigation of the role of donor-specific human leukocyte antigen antibodies (HLA-DSA) in antibody-mediated graft injury in great detail. This led to the well-evidenced paradigm that circulating HLA-DSA play a role in the pathogenesis of TG.7Hanf W. Bonder C.S. Coates P.T.H. Transplant glomerulopathy: the interaction of HLA antibodies and endothelium.J Immunol Res. 2014; 2014: 549315Crossref PubMed Scopus (17) Google Scholar, 8Cosio F.G. Gloor J.M. Sethi S. Stegall M.D. Transplant glomerulopathy.Am J Transplant. 2008; 8: 492-496Crossref PubMed Scopus (188) Google Scholar, 9Willicombe M. Brookes P. Sergeant R. et al.De novo DQ donor-specific antibodies are associated with a significant risk of antibody-mediated rejection and transplant glomerulopathy.Transplantation. 2012; 94: 172-177Crossref PubMed Scopus (166) Google Scholar, 10Issa N. Cosio F.G. Gloor J.M. et al.Transplant glomerulopathy: risk and prognosis related to anti-human leukocyte antigen class II antibody levels.Transplantation. 2008; 86: 681-685Crossref PubMed Scopus (156) Google Scholar Now, TG is classified as one of the final pathways of chronic allograft damage driven by HLA-DSA and has been incorporated into the Banff consensus criteria for the diagnosis of chronic active antibody-mediated rejection (caABMR).11Loupy A. Haas M. Roufosse C. et al.The Banff 2019 Kidney Meeting Report (I): updates on and clarification of criteria for T cell– and antibody-mediated rejection.Am J Transplant. 2020; 20: 2318-2331Crossref PubMed Scopus (90) Google Scholar For biopsies showing TG in the absence of evidence of current or recent antibody interaction with the endothelium but with a prior documented diagnosis of active ABMR or caABMR or documented prior evidence of HLA-DSA, the term chronic ABMR (cABMR) is applied.11Loupy A. Haas M. Roufosse C. et al.The Banff 2019 Kidney Meeting Report (I): updates on and clarification of criteria for T cell– and antibody-mediated rejection.Am J Transplant. 2020; 20: 2318-2331Crossref PubMed Scopus (90) Google Scholar However, there remain several gaps in our understanding and diagnosing of caABMR and cABMR. It remains unknown whether cases with TG in absence of HLA-DSA are comparable to cases with HLA-DSA–positive TG and whether these patients could also benefit from targeted antibody-removal therapies. Indeed, TG is not specific for caABMR or cABMR and can be seen in other disease processes affecting the glomerular endothelium.12Gloor J.M. Sethi S. Stegall M.D. et al.Transplant glomerulopathy: subclinical incidence and association with alloantibody.Am J Transplant. 2007; 7: 2124-2132Crossref PubMed Scopus (281) Google Scholar,13Akalin E. Dinavahi R. Dikman S. et al.Transplant glomerulopathy may occur in the absence of donor-specific antibody and C4d staining.Clin J Am Soc Nephrol. 2007; 2: 1261-1267Crossref PubMed Scopus (51) Google Scholar Several studies have suggested heterogeneity of these HLA-DSA–independent processes and reported an association among TG and hepatitis C virus infection, membranoproliferative glomerulonephritis, and chronic thrombotic microangiopathy.4Baid-Agrawal S. Farris A.B. Pascual M. et al.Overlapping pathways to transplant glomerulopathy: chronic humoral rejection, hepatitis C infection, and thrombotic microangiopathy.Kidney Int. 2011; 80: 879-885Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,14Remport A. Ivanyi B. Mathe Z. et al.Better understanding of transplant glomerulopathy secondary to chronic antibody-mediated rejection.Nephrol Dial Transplant. 2015; 30: 1825-1833Crossref PubMed Scopus (32) Google Scholar,15Haas M. Transplant glomerulopathy: it's not always about chronic rejection.Kidney Int. 2011; 80: 801-803Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar The risk factors and clinical management of patients with HLA-DSA–negative TG remain largely unknown. In this regard, 2 recent reports have suggested that the number of locus-specific HLA eplet mismatches is associated with the prediction of development TG and graft outcome, even in the absence of HLA-DSA.16Sapir-Pichhadze R. Zhang X. Ferradji A. et al.Epitopes as characterized by antibody-verified eplet mismatches determine risk of kidney transplant loss.Kidney Int. 2020; 97: 778-785Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar,17Sapir-Pichhadze R. Tinckam K. Quach K. et al.HLA-DR and -DQ eplet mismatches and transplant glomerulopathy: a nested case-control study.Am J Transplant. 2015; 15: 137-148Crossref PubMed Scopus (83) Google Scholar These findings suggested that HLA mismatch in itself may associate with an increased risk of TG development, possibly through antibody-independent processes. Although provocative, the interpretation of these studies is hampered by the lack of information on de novo HLA-DSA formation and by the use of inferred and incomplete HLA data.18Senev A. Emonds M.P. Van Sandt V. et al.Clinical importance of extended second field high-resolution HLA genotyping for kidney transplantation.Am J Transplant. 2020; 20: 3367-3378Crossref PubMed Scopus (22) Google Scholar The association of HLA molecular mismatches on the development of transplant glomerulopathy, especially in the absence of HLA-DSA, thus remains largely unclear. Therefore, we investigated the risk factors, histologic appearance, and impact of TG in a complete high-resolution HLA genotyped cohort and HLA antibody follow-up, with focus on the evaluation of TG in the absence of HLA-DSA, as this represents an ill-specified clinical phenotype. All consecutive adult single kidney transplantations performed at the University Hospitals-Leuven between March 1, 2004, and February 6, 2013, were eligible for this study. All transplantations were blood group (A, B, AB, and O)–compatible and performed with negative complement-dependent cytotoxicity. All clinical data were prospectively collected during routine clinical follow-up and stored systematically in electronic formats. This retrospective study was approved by the Ethics Committee of the University Hospitals Leuven (S53364 and S64006). All recipients and donors of this cohort with available DNA samples were genotyped retrospectively at the second field high-resolution level for 11 HLA loci—HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, and -DPB1—by next-generation sequencing. Half of the donors were genotyped using the MIA FORA NGS FLEX 11 HLA Typing Kit (Immucor Inc., Norcross, GA) on the MiSeq sequencing instrument (Illumina Inc., San Diego, CA). The other half of the donors and all recipients were genotyped at high-resolution level covering the extracellular domains of all 11 HLA molecules (exon 2, 3, and 4 for HLA class I and exon 2 and 3 for HLA class II).19Senev A. Coemans M. Lerut E. et al.Eplet mismatch load and de novo occurrence of donor-specific anti-HLA antibodies, rejection, and graft failure after kidney transplantation: an observational cohort study.J Am Soc Nephrol. 2020; 31: 2193-2204Crossref PubMed Scopus (23) Google Scholar We used the HLAMatchmaker (www.epitopes.net) and PIRCHE (Predicted Indirectly Recognizable HLA Epitopes) programs (www.pirche.com) to calculate HLA molecular mismatches.20Duquesnoy R.J. Marrari M. Tambur A.R. et al.First report on the antibody verification of HLA-DR, HLA-DQ and HLA-DP epitopes recorded in the HLA Epitope Registry.Hum Immunol. 2014; 75: 1097-1103Crossref PubMed Scopus (56) Google Scholar,21Lachmann N. Niemann M. Reinke P. et al.Donor–recipient matching based on predicted indirectly recognizable HLA epitopes independently predicts the incidence of de novo donor-specific HLA antibodies following renal transplantation.Am J Transplant. 2017; 17: 3076-3086Crossref PubMed Scopus (66) Google Scholar The second field high-resolution HLA genotypes for all 11 loci were uploaded to HLAMatchmaker (version 3.1) and PIRCHE program (version 3.1.148). From this, we obtained the eplet mismatches and PIRCHE-II scores. The pre- and post-transplant anti-HLA antibodies were screened using a LIFECODES LifeScreen Deluxe kit (Immucor) at day 0 and 3 months after transplantation, yearly after transplantation, or at the time of an indication biopsy. In a case of positive screening, HLA antibodies were identified by using LIFECODES Single Antigen Bead kits (Immucor). Anti-HLA antibodies detected in the recipient serum were analyzed for donor-specificity against HLA-A, -B, -C, -DRβ1345, -DQα1β1, and -DPα1β1 molecules. A possible presence of HLA-DSA was suspected at background-corrected median fluorescence intensity value ≥500. However, for the final assignment of HLA-DSA, as described in detail previously,18Senev A. Emonds M.P. Van Sandt V. et al.Clinical importance of extended second field high-resolution HLA genotyping for kidney transplantation.Am J Transplant. 2020; 20: 3367-3378Crossref PubMed Scopus (22) Google Scholar the sera reactivity of the patients was analyzed. At the time of an allograft biopsy, HLA-DSA positivity was determined by the persistent presence of the pretransplant HLA-DSA or documentation of de novo HLA-DSA. We included all post-transplant renal allograft biopsies performed in this cohort (N = 3744). Protocol kidney transplant biopsies were performed at the time of transplantation and at 3, 12, and 24 months after transplantation. In addition, patients who received transplants before October 1, 2005, were invited for a protocol biopsy at 36, 48, and 60 months after transplantation; patients transplanted between October 1, 2005, and November 30, 2008, for a protocol biopsy at 36 and 60 months; and patients transplanted between November 1, 2008, and January 31, 2010, for a protocol biopsy at 60 months. Scoring of the biopsies was described in detail previously.22Senev A. Coemans M. Lerut E. et al.Histological picture of antibody-mediated rejection without donor-specific anti-HLA antibodies: clinical presentation and implications for outcome.Am J Transplant. 2019; 19: 763-780Crossref PubMed Scopus (44) Google Scholar The severity of the chronic allograft glomerulopathy (cg) lesions was semi-quantitatively scored according to the Banff categories as cg0, cg1b, cg2, and cg3 based on light microscopy.23Roufosse C. Simmonds N. Clahsen-Van Groningen M. et al.A 2018 reference guide to the Banff classification of renal allograft pathology.Transplantation. 2018; 102: 1795-1814Crossref PubMed Scopus (186) Google Scholar The diagnosis of the histopathologic phenotypes of ABMR, T-cell mediated rejection (TCMR), and borderline changes was established retrospectively, according to Banff 2019 consensus and based on the individual Banff lesion scores.11Loupy A. Haas M. Roufosse C. et al.The Banff 2019 Kidney Meeting Report (I): updates on and clarification of criteria for T cell– and antibody-mediated rejection.Am J Transplant. 2020; 20: 2318-2331Crossref PubMed Scopus (90) Google Scholar Borderline changes were not included in the TCMR phenotype. Microvascular inflammation (MVI) was defined as the sum score of glomerulitis and peritubular capillaritis >1. In this article, TG was defined as a Banff lesion score cg > 0 after the exclusion of chronic thrombotic microangiopathy and recurrent or de novo glomerulonephritis or both. With lesions of disease activity, MVI ≥ 2 or MVI = 1 with C4d > 1, the biopsies with TG were classified as caABMR in the presence of HLA-DSA and as histology of caABMR (caABMRh) in the absence of HLA-DSA. Similarly, in the absence of disease activity MVI < 2 and C4d < 1, TG biopsies were classified as cABMR in the presence of HLA-DSA and as histology of cABMR (cABMRh) in the absence of HLA-DSA. The different TG archetypes, which were recently defined, were calculated according to the TG Archetype Analysis and Kidney Allograft Survival online application (https://dyshinyapps.shinyapps.io/Archetype_Shiny/).24Aubert O. Higgins S. Bouatou Y. et al.Archetype analysis identifies distinct profiles in renal transplant recipients with transplant glomerulopathy associated with allograft survival.J Am Soc Nephrol. 2019; 30: 625-639Crossref PubMed Scopus (20) Google Scholar When comparing 2 groups, Pearson chi-square test was used for categorical data (or Fisher exact test if appropriate), the 2-sample Student t test for the continuous variables, and the Wilcoxon test for the comparison of medians. Graft failure was defined as a return to dialysis or retransplantation. In case of patient death with a functioning graft, we censored graft survival at the time of death. Patients who did not experience graft failure and who were still in clinical follow-up at time of data extraction were administratively censored on September 1, 2019. Survival curves were plotted using the Kaplan-Meier method and groups were compared by log-rank testing. Univariable and multivariable Cox proportional hazards models were applied to quantify the hazard ratios (HRs) of transplant characteristics for developing TG or kidney allograft failure. When TG was used as endpoint in survival analyses, the last biopsy performed per patient was considered as the end of follow-up. Finally, landmark analyses were performed at 1 and 3 years post-transplant. All P values <0.05 were considered to indicate statistical significance. We used SAS (version 9.4; SAS Institute, Cary, NC) and GraphPad Prism software (version 9.0; GraphPad Software, San Diego, CA) for the statistical analyses. The study cohort contained 954 single kidney transplant recipients with available allograft biopsy follow-up (Figure 1). The median follow-up time post-transplant of this cohort was 7.99 (interquartile range [IQR]: 5.21–10.6) years (Supplementary Table S1). Among these patients, we identified 105 cases with at least 1 biopsy with a Banff cg score >0 on light microscopy. TG was diagnosed in 98 of 954 patients (10.3%), according to the Banff 2019 criteria, on average at 3.16 ± 2.81 years post-transplant.11Loupy A. Haas M. Roufosse C. et al.The Banff 2019 Kidney Meeting Report (I): updates on and clarification of criteria for T cell– and antibody-mediated rejection.Am J Transplant. 2020; 20: 2318-2331Crossref PubMed Scopus (90) Google Scholar An additional 7 patients had cg > 0 with chronic thrombotic microangiopathy or membranoproliferative glomerulonephritis and therefore were not included as TG in this article.23Roufosse C. Simmonds N. Clahsen-Van Groningen M. et al.A 2018 reference guide to the Banff classification of renal allograft pathology.Transplantation. 2018; 102: 1795-1814Crossref PubMed Scopus (186) Google Scholar The remaining 849 patients did not have TG (cg = 0) on light microscopy in any of their biopsies performed during the histologic follow-up (n = 3243). The cumulative incidence of TG in this cohort of 954 kidney transplant recipients at 1, 2, 3, and 5 years after transplantation was 3.42%, 8.05%, 10.4%, and 17.2%, respectively (Figure 2a). In the univariable Cox analysis, repeat kidney transplantation, living donation, cold ischemia time, induction therapy at transplantation, and pretransplant HLA-DSA were associated with an increased risk of TG development (Table 1). We found no significant association between pretransplant non–HLA-DSA, HLA antigen mismatches, eplet mismatches, and PIRCHE-II score with the development of TG. In a consequent multivariable Cox model, including all significant predictors of the univariable analysis, only pretransplant HLA-DSA remained as an independent risk factor for developing TG (HR: 3.00; 95% confidence interval [CI]: 1.74–5.18; P < 0.0001).Table 1Univariable and multivariable Cox proportional hazards analyses of the association between pre-Tx factors and the development of TGPredictorNo. of patients (n = 954)No. of events (n = 98)HR95% CIP valueUnivariable analysisPrimary glomerular disease (yes vs. no)272280.880.57–1.380.58Repeat kidney Tx (yes vs. no)145211.981.22–3.230.006Recipient sex (male vs. female)584530.710.48–1.070.10Recipient age (by 10 yr)954981.010.86–1.180.92Recipient body mass index (by 1 unit)954980.970.92–1.010.14Donor sex (male vs. female)509540.930.62–1.390.72Donor age (by 10 yr)954981.120.97–1.290.14Type of donation (living vs. deceased)57111.931.03–3.620.04Cold ischemia time (per 1 h)954980.970.94–1.000.05Immunosuppression (TAC-MPA-CS vs. other)814841.340.76–2.360.32Induction therapy at Tx (yes vs. no)396541.831.22–2.730.003Pre-TX anti-HLA antibodies (yes vs. no)250412.401.60–3.59<0.0001Pre-TX anti-HLA non-DSA antibodies (yes vs. no)144171.260.75–2.140.37Pre-TX HLA-DSA (yes vs. no)106243.302.08–5.24<0.0001HLA-A/B/C/DR antigen MM (per 1 MM)954981.020.87–1.190.86HLA-A/B/C/DR/DQ antigen MM (per 1 MM)893921.030.90–1.170.68Total HLA eplet mismatches (per 10 MM eplets)aA total of 61 transplant pairs were excluded from this analysis due to the lack of complete second field HLA typing data for all 11 HLA loci.893921.020.87–1.190.85HLA class I eplet MM load (per 10 MM eplets)893920.920.65–1.310.64HLA class II eplet MM load (per 10 MM eplets)893921.050.87–1.260.63DRB1345/DQA1B1 eplet MM load (per 10 MM eplets)893921.060.87–1.280.57DRB1345 eplet MM load (per 10 MM eplets)893921.080.78–1.510.64DQA1B1 eplet MM load (per 10 MM eplets)893921.080.79–1.490.62PIRCHE-II score (per 10)aA total of 61 transplant pairs were excluded from this analysis due to the lack of complete second field HLA typing data for all 11 HLA loci.893921.000.99–1.010.70PIRCHE-II score for HLA class I (per 10)893920.990.97–1.020.59PIRCHE-II score for HLA class II (per 10)893921.010.99–1.020.41PIRCHE-II score for DRB1345 + DQA1B1 (per 10)893921.010.99–1.020.49PIRCHE-II score for DRB1345 (per 10)893921.000.97–1.040.99PIRCHE-II score for DQA1B1 (per 10)893921.010.99–1.030.33Multivariable analysisPre-TX HLA-DSA (yes vs. no)106243.011.74–5.19<0.0001Repeat kidney Tx (yes vs. no)145211.030.57–1.850.93Type of donation (living vs deceased)57111.110.42–2.950.83Cold ischemia time (per 1 h)954980.980.93–1.030.34Induction therapy at Tx (yes vs. no)396981.510.98–2.320.06CI, confidence interval; CS, corticosteroids; DSA, donor-specific antibodies; HLA, human leukocyte antigen; HR, hazard ratio; HLA-DSA, donor-specific HLA antibodies; MM, mismatch; MPA, mycophenolic acid; PIRCHE, Predicted Indirectly Recognizable HLA Epitopes; TAC, tacrolimus; TG, transplant glomerulopathy; TX, transplantation.Patients in this analysis were censored at the date of the last performed biopsy (n = 954). Statistically significant differences are highlighted in bold.a A total of 61 transplant pairs were excluded from this analysis due to the lack of complete second field HLA typing data for all 11 HLA loci. Open table in a new tab CI, confidence interval; CS, corticosteroids; DSA, donor-specific antibodies; HLA, human leukocyte antigen; HR, hazard ratio; HLA-DSA, donor-specific HLA antibodies; MM, mismatch; MPA, mycophenolic acid; PIRCHE, Predicted Indirectly Recognizable HLA Epitopes; TAC, tacrolimus; TG, transplant glomerulopathy; TX, transplantation. Patients in this analysis were censored at the date of the last performed biopsy (n = 954). Statistically significant differences are highlighted in bold. The cumulative incidence of TG at 5 years after transplantation was significantly higher in the patients with pretransplant HLA-DSA compared with patients who are HLA-DSA–negative pretransplant (38.3% vs. 15.0%; P < 0.0001) (Figure 2b). However, of the 98 patients with TG, only 23 (23.5%) had HLA-DSA at the time of TG biopsy, mainly class II (21 of 23; 91.3%), and 75 (76.5%) were HLA-DSA–negative (Figure 1). Next, as the TG phenotype was the more prevalent in the absence than in the presence of HLA-DSA, we investigated the main predictors of developing TG in patients who are HLA-DSA–negative pretransplant (n = 848), censored at the time of de novo HLA-DSA occurrence (Table 2). In this univariable Cox analysis, recipient sex (male vs. female; HR: 0.61; 95% CI: 0.37–1.01; P = 0.05) and donor age (HR: 1.26 per 10-year increment; 95% CI: 1.04–1.52; P = 0.02) associated with the development of TG in the absence of HLA-DSA. HLA molecular mismatches, assessed as total eplet mismatches (HR: 1.00; 95% CI: 0.83–1.20; P = 0.98) and PIRCHE-II score (HR: 1.00; 95% CI: 0.99–1.01; P = 1.00), were not associated with an increased risk of developing TG in the absence of HLA-DSA. Older donor age remained as the only independent risk factor for developing TG in the absence of HLA-DSA in multivariable analysis (HR: 1.22 per 10 years; 95% CI: 1.03–1.46; P = 0.02).Table 2Univariable and multivariable Cox proportional hazards analyses of the association between pre-Tx factors and the development of TG in the pre-TX HLA-DSA–negative cohortPredictorNo. of patients (n = 848)No. of events (n = 65)HR95% CIP valueUnivariable analysisPrimary glomerular disease (yes vs. no)241180.880.51–1.520.66Repeat kidney Tx (yes vs. no)8681.710.81–3.610.09Recipient sex (male vs. female)539350.610.37–1.010.05Recipient age (by 10 y)848651.140.93–1.390.20Recipient body mass index (kg/m2) (per 1 unit)848650.980.93–1.040.50Donor sex (male vs. female)455360.910.55–1.490.91Donor age (by 10 yr)848651.261.04–1.520.02Type of donation (living vs. deceased)4961.510.65–3.520.34Cold ischemia time (per 1 h)848650.970.93–1.010.18Immunosuppression (TAC-MPA-CS vs. other)717561.490.73–3.020.27Induction therapy at Tx (yes vs. no)324281.210.74–1.980.45Pre-Tx HLA non-DSA antibodies (yes vs. no)144151.630.91–2.910.10HLA-A/B/C/DR mismatches (per 1 MM)848651.020.84–1.230.87HLA-A/B/C/DR/DQ mismatches (per 1 MM)798621.000.86–1.171.00Total HLA eplet MM load (per 10 MM eplets)aA total of 50 transplant pairs were excluded from this analysis due to the lack of complete second field HLA typing data for all 11 HLA loci.798621.000.83–1.200.98HLA class I eplet MM load (per 10 MM eplets)798621.170.77–1.790.47HLA class II eplet MM load (per 10 MM eplets)798620.950.76–1.200.68HLA DRB1345/DQA1B1 eplet MM load (per 10 MM eplets)798620.960.76–1.220.73HLA DRB1345 eplet MM load (per 10 MM eplets)798620.940.62–1.430.79HLA-DQA1B1 eplet MM load (per 10 MM eplets)798620.940.63–1.390.75PIRCHE-II score (per 10)aA total of 50 transplant pairs were excluded from this analysis due to the lack of complete second field HLA typing data for all 11 HLA loci.798621.000.99–1.011.00PIRCHE-II score for HLA class I (per 10)798621.000.98–1.030.82PIRCHE-II score for HLA class II (per 10)798621.000.98–1.020.88PIRCHE-II score for DRB1345 + DQA1B1 (per 10)798621.000.98–1.020.84PIRCHE-II score for DRB1345 (per 10)798620.990.95–1.040.77PIRCHE-II score for DQA1B1 (per 10)798621.000.97–1.030.91Multivariable analysisDonor age (by 10 yr)848651.251.04–1.510.02Recipient sex (male vs. female)539350.630.38–1.030.07CI, confidence interval; CS, corticosteroids; DSA, donor-specific antibodies; HLA, human leukocyte antigen; HR, hazard ratio; HLA-DSA, donor-specific HLA antibodies; MM, mismatch; MPA, mycophenolic acid; PIRCHE, Predicted Indirectly Recognizable HLA Epitopes; TAC, tacrolimus; TG, transplant glomerulopathy; TX, transplantation.Patients in this analysis were censored at the date of de novo HLA-DSA occurrence or at the last performed biopsy (n = 848). Statistically significant differences are highlighted in bold.a A total of 50 transplant pairs were excluded from this analysis due to the lack of complete second field HLA typing data for all 11 HLA loci. Open table in a new tab CI, confidence interval; CS, corticosteroids; DSA, donor-specific antibodies; HLA, human leukocyte antigen; HR, hazard ratio; HLA-DSA, donor-specific HLA antibodies; MM, mismat