CLINICAL RESEARCH www.jasn.org Early Acute Microvascular Kidney Transplant Rejection in the Absence of Anti-HLA Antibodies Is Associated with Preformed IgG Antibodies against Diverse Glomerular Endothelial Cell Antigens Marianne Delville,1,2,3 Baptiste Lamarthée,4 Sylvain Pagie,5,6 Sarah B. See ,7 Marion Rabant,3,8 Carole Burger,3 Philippe Gatault ,9,10 Magali Giral,11 Olivier Thaunat,12,13,14 Nadia Arzouk,15 Alexandre Hertig,16,17 Marc Hazzan,18,19,20 Marie Matignon,21,22,23 Christophe Mariat,24,25 Sophie Caillard,26,27 Nassim Kamar,28,29 Johnny Sayegh,30,31 Pierre-François Westeel,32 Cyril Garrouste,33 Marc Ladrière,34 Vincent Vuiblet,35 Joseph Rivalan,36 Pierre Merville,37,38,39 Dominique Bertrand,40 Alain Le Moine,41,42 Jean Paul Duong Van Huyen,3,8 Anne Cesbron,43 Nicolas Cagnard,3,44 Olivier Alibeu,3,45 Simon C. Satchell,46 Christophe Legendre,3,4,47 Emmanuel Zorn,7 Jean-Luc Taupin,48,49,50 Béatrice Charreau,5,6 and Dany Anglicheau 3,4,47 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allo- grafts, non-anti–HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed. Methods We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combi- nation of transcriptomic and proteomic approaches to identify non-HLA Abs. Results We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti–endothelial cell Abs—angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural poly- reactive Abs—did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Tran- scriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals. Conclusions Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that in vitro cell-based assays are needed to improve risk assessments before transplant. J Am Soc Nephrol 30: 692–709, 2019. doi: https://doi.org/10.1681/ASN.2018080868 Despite the development of potent immunosup- suggestive of AMR (i.e., microvascular inflamma- pressive regimens, antibody-mediated rejection tion) usually indicate an anti-HLA–mediated (AMR) remains a significant hurdle to long-term injury, a subset of patients develop these lesions in organ acceptance. Although histologic findings the absence of detectable anti-HLA donor-specific 692 ISSN : 1046-6673/3004-692 J Am Soc Nephrol 30: 692–709, 2019 www.jasn.org CLINICAL RESEARCH antibodies (DSAs). The potential involvement of non-HLA an- Significance Statement tibodies (Abs) is mentioned in the current Banff classification, which requires the presence of “serological evidence of DSAs Antibody-mediated rejection (AMR) in renal allografts, which is against HLA or other antigens.”1 However, in the absence of usually caused by antibodies (Abs) directed against HLAs, is asso- fi ciated with a poor transplant outcome. However, evidence of AMR other, clearly de ned antigens, the assumption that acute re- – fi fl in the absence of anti-HLA Abs suggests the presence of non-anti jections with signi cant microvascular in ammation (called HLA Abs, presumed to react with other antigens on endothelial acute microvascular rejections [AMVRs]) are true AMRs re- cells. The authors describe the clinicopathologic profiles of kid- mains hypothetical. In addition, although this issue is of ut- ney recipients who experienced acute rejection with microvascular most importance for treatment decisions, a clear indication inflammation within 3 months after transplantation in the absence fi that the observed graft injury is induced by Abs may be difficult of anti-HLA donor-speci c Abs. Using a new endothelial cell crossmatch assay and transcriptomic and proteomic analyses, they to obtain. discovered that before transplantation, these patients carried un- These particular types of immune injuries are presumed to known anti–endothelial cell Abs in their sera that specifically tar- be because of Abs that react with non-HLA antigens expressed geted the glomerular microvascular endothelium. An assessment of on endothelial cells (ECs). These Abs might be alloantibodies these unknown potentially deleterious Abs may provide important directed against non-HLA polymorphic antigens that differ diagnostic tools to prevent AMR. between the recipient and donor or autoantibodies that rec- ognize self-antigens after a disruption of self-tolerance.2 The AMVRs of renal allografts in the absence of anti-HLA identification and characterization of pathogenic anti– DSAs. Inclusion criteria were a first transplantation or endothelial cell Abs (AECAs) would improve our understand- retransplantation, a deceased or living donor, acute dysfunc- ing of the mechanisms involved in AMRs and would enable tion or delayed graft function occurring within the first 3 the development of new tools for patient monitoring. Several months post-transplantation, histologic features of microvas- hurdles hamper the identification of these AECAs. First, the cular inflammation with a glomerulitis plus peritubular capil- development of acute renal dysfunction with histologic lesions laritis score $3 according to the Banff classification, and the suggestive of AMR in the absence of anti-HLA DSAs is a rel- absence of historical or current anti-HLA DSA (A/B/Cw/DR/ atively rare event. Consequently, previous studies that aimed DQ/DP), as assessed using a Luminex single-antigen bead to identify AECAs often included patients with heterogeneous assay (all mean fluorescence intensities ,500). All biopsy clinical presentations ranging from hyperacute rejection3–5 to specimens were centrally reassessed, and the absence of anti- chronic allograft dysfunction,5,6 or patients with a positive EC HLA DSAs was also centrally confirmed (see Supplemental crossmatch independent of any clinical presentation.7 Second, Material for details). the identification of deleterious non-HLA Abs is particularly For the case-control histologic study (Figure 1), a control difficult to achieve in long-term patients, as a broad autoan- group of 20 KTRs with early full-blown AMR who presented tibody response develops over time after transplantation.8,9 with anti-HLA DSAs in the first 3 months was identified. The We aimed to study a highly selected cohort of patients with patients were matched for age, sex, time of transplantation, a homogeneous clinical and pathologic presentation and immunosuppressive regimen at transplantation. of AMVR without anti-HLA DSAs during the first 3 months For the case-control biologic study (Figure 1), a second post-transplantation, to overcome these challenges. Wereasoned control group of ten highly stable patients (i.e., no rejection that early AMVR would likely be caused by preformed AECAs, during the first year) was identified. Patients in this control facilitating their identification in pretransplant serum samples. group were also matched to patients in the AMVR group for We report here the clinicopathologic description of this cohort age, sex, time of transplantation, and immunosuppressive reg- and our efforts to identify the pathogenic AECAs. imen at transplantation. METHODS Non-HLA Antibody Detection Methods for the detection of non-HLA Abs, including anti- Patients MICA, anti-AT1R, anti-ETAR, natural Abs, and Abs against a Kidney transplant recipients (KTRs) were identified through a panel of 62 non-HLA antigens, in patients’sera are described in nationwide survey aimed at identifying suspected cases of early detail in the Supplemental Material. Received August 25, 2018. Accepted January 31, 2019. EC Crossmatch Different types of ECs were incubated with patients’ serum M.D. and B.L. contributed equally to this work. samples, and IgG fixation was detected using flow cytometry. Published online ahead of print. Publication date available at www.jasn.org. A comparative analysis of the reactivity of the patient’sserum Correspondence: Prof. Dany Anglicheau, Service de Néphrologie et Trans- was performed on parallel crossmatches using primary cul- plantation Rénale Adulte, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, tures of non-donor-specific arterial ECs and the immortalized 75015 Paris, France. Email: [email protected] human glomerular microvascular EC line CiGEnC (see the Copyright © 2019 by the American Society of Nephrology Supplemental Material for details). J Am Soc Nephrol 30: 692–709, 2019 Anti-Endothelial Cell Antigens in Renal Microvascular Rejection 693 CLINICAL RESEARCH www.jasn.org RNA Sequencing and Protein Array 3 months (161659 mmol/L versus 129655 mmol/L; P=0.01), RNA sequencing (RNAseq) was performed to assess the differ- and similar graft function at the last follow-up (P=0.23). Con- ences in the transcriptomes between microvascular and macro- sistent with severe graft injury, proteinuria was common in vascular ECs. Patients’ serum samples were applied to a protein both groups, and after a similar follow-up period, the protein- array to assess the seroreactivity of stable KTRs and patients with uria in the AMVR cohort was similar to the AMR cohort AMVR (see the Supplemental Material for details). (1.2761.7 g/g versus 1.061.4 g/g; P=0.44).