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1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Lefaucheur C, Loupy A, Vernerey D, et al. Antibody-mediated vascular rejection of kidney allografts: a population-based study. Lancet 2012; published online Nov 23.

2 SUPPLEMENTARY MATERIAL Supplementary methods 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 agency database CRISTAL (Official website: DIVAT and CRISTAL databases network have been 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 3th April Codes were used to ensure the strict donor and recipient anonymity and blind assay. Informed consent is obtained from the participants at time of transplantation. The data are computerized in real time as well as at each transplant anniversary and are submitted for an annual audit. Independent validation cohort This external validation set is comprised of the 672 consecutive kidney recipients transplanted in Foch Hospital, Suresnes, France. Kidney transplantations were performed between 1 st January, 2004 and 31 January, The median follow-up post transplant was 36.4 months. Similar immunological rules for kidney transplantation applied, with all transplantations being ABO compatible with a negative current IgG T-cell and B-cell complement-dependent cytotoxicity cross-matching required for all patients. Patients with primary nonfunctioning grafts due to surgical vascular or urologic issues were also excluded from the analysis (n=11). Among the 661 kidney allograft recipients included, we identified 133 patients (20.1%) with a diagnosis of acute clinical biopsy-proven rejection. All biopsies were graded according to the updated Banff criteria 7,8 by a trained pathologist. The C4d staining was performed by immunochemistry according to the same protocol used in Necker and Saint Louis Hospitals (Biomedica Gruppe, Austria). Detection of antibodies against donor-specific HLA molecules was centralized in the regional histocompatibility laboratory. 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. Subsequent maintenance immunosuppressive therapy consisted 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 after 3 months or cyclosporine administered to maintain a target blood level of 800 to 1200 ng per milliliter for the first 3 months and 600 to 800 ng per milliliter after 3 months. Patients with preexisting donor- specific anti- HLA antibodies, as detected by the techniques in current use at the time of transplantation, received intravenous immune globulin (2 g per kilogram of body weight on days 0, day 20 and day 40) ± rituximab given twice (375 mg per square meter of body- surface area on day 0 and day 7) as prophylaxis against acute rejection according to center practice 1

3 Supplementary Table Supplementary table 1: Interpretation of Principal Component Analysis axis shown in Figure 1. Supplementary Figures Supplementary Figure 1: Individual Overview of Morphological and Immunological Rejection Profiles according to Unsupervised Cluster Analysis. Supplementary Figure 2: Histologic and Immunohistochemical Lesions of Antibody-Mediated Vascular Rejection. Supplementary Figure 3: Kaplan-Meier Curves for 6-Years Kidney Graft Survival in Antibody-Mediated Vascular Rejection according to the initial Diagnosis made by the Classical Banff Approach and by the New Approach. Supplementary Figure 4: Validation cohort: Unsupervised Principal Component Analysis (PCA) in Patients with Acute Rejection of Kidney Allograft. Supplementary Figure 5: Kaplan-Meier Curves for kidney allograft Survival by Acute Rejection phenotype in the validation cohort (N=661). 2

4 Supplementary Table 1: The 4 rejection patterns described in figure 1 are segregated according to 2 axes. The horizontal axis opposes cellular rejection (interstitial inflammation and tubulitis) with antibodymediated rejection (donor-specific anti-hla antibodies, glomerulitis, peritubular capillaritis and C4d) as recognized by the current international Banff classification. The vertical axis defines the presence or absence of lesions of endarteritis. Relative contributions of variables (%) Horizontal axis 1 Vertical axis 2 Endarteritis 2 50 Interstitial inflammation Tubulitis C4d 16 0 Peritubular capillaritis 17 8 Donor-specific anti-hla antibodies 19 1 Glomerulitis : Horizontal axis opposes T cell mediated rejection to antibody-mediated rejection (as recognized by the current Banff classification) 2 : Vertical axis defines the presence or absence of lesions of endarteritis 3

5 Supplementary Figure 1: Individual Overview of Morphological and Immunological Rejection Profiles according to Unsupervised Cluster Analysis. Each variable in an individual patient is colored according to threshold as defined by Banff classification. DSA denotes donor-specific anti-hla antibodies levels according to thresholds as previously defined 4

6 Supplementary Figure 2: Histologic and Immunohistochemical Lesions of Antibody-Mediated Vascular Rejection A Endarteritis with prominent intimal inflammation (v2). B- Transmural arteritis with necrosis (v3). C- Antibody mediated endarteritis with prominent macrophage, and peritubular capillary leukcytic infiltration (arrows). D- Biopsy in a C4d positive patient, stained for C4d. Artery (top) with diffuse intimal staining for C4d and early endarteritis lesion (arrows). Peritubular capillaries stain diffusely (Magnification X375). 5

7 Supplementary Figure 3: Kaplan-Meier Curves for 6-Years Kidney Graft Survival in Antibody-Mediated Vascular Rejection according to the initial Diagnosis made by the Classical Banff Approach and by the New Approach. New approach (n=22) Traditional approach (n=42) Graft survival (%) Log rank: p= Time post rejection (months) Number at risk

8 Supplementary Figure 4: Validation cohort: Unsupervised Principal Component Analysis (PCA) in Patients with Acute Rejection of Kidney Allograft. The PCA analysis of the 133 patients of the validation set with acute biopsy-proven rejection using 7 variables: glomerulitis, interstitial inflammation, tubulitis, endarteritis, peritubular capillaritis, donorspecific anti-hla antibodies and C4d. The 4 rejection patterns described in figure 1 are segregated according to 2 axes. The horizontal axis opposes Glomerulitis, donor-specific anti-hla antibodies, PTC and C4d (ABMR V-; ABMR V+) to interstitial inflammation and tubulitis (TCMR/V-; TCMR V+) while the vertical PC axis is principally linked with endarteritis parameters. TCMR/V- denotes T cell-mediated rejection without vasculitis, ABMR/V- Antibody-mediated rejection without vasculitis, TCMR/V+ T cell-mediated vascular rejection and ABMR/V+ Antibody-mediated vascular rejection. 7

9 Supplementary Figure 5: Kaplan-Meier Curves for kidney allograft Survival by Acute Rejection phenotype in the validation cohort (N=661). No Rejection (n=528)* TCMR/V- (n=34) TCMR/V+ (n=15) ABMR/V-(n=57) ABMR/V+(n=27) Graft survival (%) Number at risk Log rank: p< Time post Rejection (months) * No rejection * TCMR/V- TCMR/V+ ABMR/V- ABMR/V * Graft survival in patients without rejection is purely illustrative. In this situation, graft survival starts at time of transplantation 8

Supplementary Appendix

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