Supplementary Appendix

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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Loupy A, Lefaucheur C, Vernerey D, et al. Complement-binding anti-hla antibodies and kidneyallograft survival. N Engl J Med 2013;369: DOI: /NEJMoa

2 Supplementary appendix Table of contents: Author s contributions (Page 2) Supplementary methods (Page 3 6) Supplementary Figures (Page 7 12) Supplementary Tables (Page 13 14) 1

3 AUTHORS CONTRIBUTIONS - Study design: Alexandre Loupy, Carmen Lefaucheur. - Data collection: Alexandre Loupy, Carmen Lefaucheur, Jean-Paul Duong van Huyen, Nuala Mooney, Caroline Suberbielle, Arnaud Méjean, François Desgrandchamps, Dominique Nochy, Dany Anglicheau, Dominique Charron, Michel Delahousse, Christophe Legendre, Denis Glotz, Gary S. Hill, Adriana Zeevi. - Data gathering: Dewi Vernerey, Alexandre Loupy, Carmen Lefaucheur, Jean Philippe Empana, Xavier Jouven. - Data analysis: Alexandre Loupy, Carmen Lefaucheur, Dewi Vernerey Jean Philippe Empana, Christof Prugger, Xavier Jouven. - Data vouching: Alexandre Loupy, Carmen Lefaucheur, D Vernerey, Jean-Paul Duong van Huyen, Nuala Mooney, Caroline Suberbielle, Arnaud Méjean, François Desgrandchamps, Dany Anglicheau, Dominique Charron, Michel Delahousse, Jean Philippe Empana, Véronique Frémeaux-Bacchi, Christophe Legendre, Denis Glotz, Gary S. Hill, Adriana Zeevi, Xavier Jouven. - Manuscript writing: Alexandre Loupy, Carmen Lefaucheur, D Vernerey, Jean Philippe Empana, Christof Prugger, Christophe Legendre, Denis Glotz, Gary S. Hill, Adriana Zeevi, Xavier Jouven. - Final approval of the version to be published: Alexandre Loupy, Carmen Lefaucheur, D Vernerey, Christof Prugger, Jean-Paul Duong van Huyen, Nuala Mooney, Caroline Suberbielle, Arnaud Méjean, François Desgrandchamps, Dany Anglicheau, Dominique Charron, Michel Delahousse, Jean Philippe Empana, Dominique Nochy, Véronique Frémeaux- Bacchi, Christophe Legendre, Denis Glotz, Gary S. Hill, Adriana Zeevi, Xavier Jouven. - Writing of the first draft of the manuscript: Alexandre Loupy & Carmen Lefaucheur. - The authors certify that neither this article nor any part of its essential substance, tables or figures has been or will be published or submitted elsewhere. All authors have seen and approved the final text of the manuscript. 2

4 SUPPLEMENTARY METHODS Post-transplantation induction protocols and maintenance of immunosuppressive therapy All patients received induction therapy consisting of rabbit antithymocyte globulin (1.5 mg per kilogram per day, for 10 days) or basiliximab (20 mg at day 0 and day 4) immediately after transplantation. All sensitized patients received induction by rabbit antithymocyte globulin. In addition, patients considered at the highest immunological risk (Preformed DSA by Luminex assay and cytotoxic reactivity (historical positive CXM or PRA > 20%) received supplementary immunosuppression at time of transplantation 24, 40 : In the Necker center 38 patients received prophylactic high dose intravenous immunoglobulin courses, 5 plasma exchange sessions and Rituximab (375 mg per square meter of body-surface area). In Saint Louis Hospital, 21 patients received prophylactic high dose intravenous immunoglobulin. In both centers, intravenous immunoglobulin was administered at a dose of 2g/kg BW over a 72-hour period of time. The first IVIg course was started before reperfusion, with subsequent courses being given on days 21, 42 and 63 after kidney transplantation. Subsequent maintenance immunosuppressive protocols were similar between centers consisting of prednisone, mycophenolate mofetil (1000 mg twice daily), tacrolimus administered to maintain a target blood level of 8 to 10 ng per milliliter for the first 3 months and 6 to 8 ng per milliliter thereafter or cyclosporine administered to maintain a target C2 blood level of 800 to 1200 ng per milliliter for the first 3 months and 600 to 800 ng per milliliter thereafter. 3

5 Treatment of allograft-rejection episodes Patients with rejection episodes diagnosed as T-cell-mediated were treated with methylprednisolone pulses (500 mg/d for 3 days). Patients with rejection uncontrolled by this regimen received additional rabbit antithymocyte globulin (1.5 mg per kilogram per day, for 5 days) or muromonab-cd3 (5 mg/d for 5 days). Patients who had episodes of antibody-mediated rejection were initially given methylprednisolone pulses (500 mg per day for 3 days), intravenous immune globulin (2 g per kilogram, repeated every three weeks for 4 rounds), four plasmaphereses and two weekly doses of rituximab (375 mg per square meter of body-surface area) following the recommendation at the French society for organ transplant established in January Data collection procedures All data from Necker Hospital regarding donor and recipient were extracted from the DIVAT clinical prospective cohort (Official website: Data from Saint Louis and Foch Hospitals were excerpted from the French national registry agency (Agence de la Biomédecine) database CRISTAL (Official website: Each patient from the present study is given written informed consent to be included in the DIVAT and CRISTAL database networks. These registries are approved by the National French Commission for bioinformatics data and patients liberty: DIVAT: CNIL, Registration number: , validated 8th June 2004 and CRISTAL: CNIL, Registration number: , validated 3rd April Codes were used to ensure the strict donor and recipient anonymity and blind assay. The data are computerized in real time as well as at each transplant anniversary and are submitted for an annual audit. The study was approved by the Institutional Review Boards of Necker Hospital, Saint-Louis Hospital and Foch Hospital without requirement for supplementary written informed consent. We excluded patients with primary non-functioning grafts due to surgical vascular or urologic problems, patients without biopsies performed in the first year post transplant and patients without adequate material to determine anti-hla complement binding capacity (n=114). 4

6 Primary kidney disease The primary kidney disease was diabetic nephropathy in 101 patients (9.9%), vascular nephropathy in 82 patients (8.1%), glomerulopathy in 267 patients (26.3%), congenital nephropathy in 185 patients (18.2%), interstitial nephropathy in 120 patients (11.8%), 48 (4.7%) patients had other disease and 213 (21.0%) patients in whom the primary kidney disease was not determined. There was no difference in term of primary kidney disease repartition within the 3 groups of patients (i.e. patients with post-transplant complementbinding donor-specific anti-hla antibodies, patients with non-complement-binding donorspecific anti-hla antibodies and patients without donor-specific anti-hla antibodies respectively, p=0.9923) Graft histology and immunochemistry All graft biopsies were scored and graded from 0 to 3 according to the updated Banff criteria 22, 23 for the following histological factors: glomerular inflammation (glomerulitis), tubulitis, interstitial inflammation, endarteritis, peritubular capillary inflammation (capillaritis), transplant glomerulopathy, interstitial fibrosis, tubular atrophy and arteriosclerosis. The microcirculation inflammation score was defined as the sum of glomerular and peritubular capillary inflammation, and the tubular and interstitial score by the sum of interstitial inflammation and tubulitis. Donor-specific anti-hla antibodies evaluation We used banked serum samples from transplant patients centralized for the 3 participating centers at the Jean Dausset histocompatibility laboratory. All beads showing a normalized mean fluorescence intensity (MFI) higher than 500 were considered positive as previously described 24. For each patient we recorded the number, specificity and the MFI of all DSA also detected. The maximum MFI of DSA was defined as the highest ranked donor-specific bead. 5

7 HLA typing of donors and recipients was performed by molecular biology (Innolipa HLA typing kit, Innogenetics, Belgium). For all kidney transplant donors, tissue-typing was done using the microlymphocytotoxicity technique with One Lambda INC tissue-typing trays at time of transplantation and reevaluated by molecular biology for this study. Detection of complement binding anti-hla antibodies Briefly, heat inactivated serum (56 C for 30 minutes) was spiked with 150 mg/ml purified human C1q in HEPES buffer (One Lambda) to ensure equal functional amounts of C1q per sample. Single antigen beads were added to the mixture and incubated for 20 minutes at room temperature, followed by addition of phycoerythrin conjugated anti-human C1q. Beads were washed twice and analyzed on a LABScan200 flow analyzer. The negative background values in C1q assay ranged from 0 to 30 MFI with most less than 10 MFI. Antibodies were assigned as possible at >300 MFI and as positive at >500 MFI that represented greater than 50% over background. 6

8 Figure S1: Kidney allograft injury phenoptype according to post transplant anti-hla antibody status. A: 1-year protocol biopsies (n=845), B: Acute rejection biopsies (n=171) A 1-YEAR PROTOCOL BIOPSIES (n=845) Microcirculation inflammation C4d graft deposition p< p< % of patients p< p< Transplant glomerulopathy Interstitial inflammation Tubulitis 0.5 p= p= p= Interstitial fibrosis Tubular atrophy 2.0 Arteriosclerosis

9 B ACUTE REJECTION BIOPSIES (n=171) Microcirculation inflammation C4d graft deposition 4 p< p< p< % of patients p= Transplant glomerulopathy Interstitial inflammation Tubulitis p= p< Interstitial fibrosis Tubular atrophy Arteriosclerosis

10 Figure S2: Kaplan Meier Analysis of graft outcome according to post-transplant DSA-MFI and complement-binding status 9

11 Figure S3: Kaplan Meier Curves of graft survival by post-transplant complement-binding DSA status and study center. DSA+ C1q-: patients with non-complement-binding DSA DSA+ C1q+: patients with complement-binding DSA 10

12 Figure S4: Incidence of kidney allograft loss according to DSA C1q and subcategory of glomerular filtration rate GFR: Glomerular filtration rate estimated by Modification of Diet in Renal Disease (MDRD equation) C1q-: patients with non-complement-binding DSA C1q+: patients with complement-binding DSA 11

13 Figure S5: Kaplan-Meier Curves for 5-Years Kidney Graft Survival by posttransplant DSA status in the external validation cohort (N=643). DSA-: patients without circulating DSA DSA+ C1q-: patients with non-complement-binding DSA DSA+ C1q+: patients with complement-binding DSA 12

14 Table S1: Combined associations of MFI and post-transplant complement-binding status with kidney graft loss in patients with post-transplant DSA Number of patients Number of events HR 95%CI p Post-transplant MFI DSA < [0.779 ; 4.383] Post-transplant Complement binding DSA [0.659 ; 4.590] No Yes [2.233 ; 8.983] <.0001 MFI: Mean Fluorescence intensity DSA: Donor-specific antibodies 13

15 Table S2: Baseline characteristics of donors and recipients in the validation cohort (n=643) and in the principal cohort (n=1016). Patients Characteristics External validation cohort (N=643) N Development cohort (N=1016) N p Recipient age years ± ± 13 < Recipient male sex no. (%) β (57%) (59%) NS Retransplantation no. (%) β (8%) (18%) < Time since dialysis years ± ± 4.7 < Donor age (years) ± ± Donor gender male no. (%) β (54%) (55%) NS Deceased donor no. (%) β (85%) (82%) NS Cold ischemia time (hours) ± ± 9.6 NS Primary kidney disease Diabetes no. (%) 56 (8%) 101 (9.9%) Vascular no. (%) 49 (8%) 82 (8.1%) Glomerulopathy no. (%) 143 (22%) 267 (26.3%) Congenital no. (%) 23 (4%) 185 (18.2%) Other no. (%) 48 (8%) 48 (4.7%) Interstitial nephropathy no. (%) 152 (24%) 120 (11.8%) Undetermined no. (%) 172 (27%) 213 (21.0%) Immunological characteristics HLA A/B/DR mismatch ± ± 1.5 < Recipient Blood group type β A/B/O/AB no /58/269/ /94/424/37 NS 14