Endothelial Cell Antibodies Associated with Novel Targets and Increased Rejection

BASIC RESEARCH www.jasn.org

† ‡ † † Annette M. Jackson,* Tara K. Sigdel, Marianne Delville, Szu-Chuan Hsieh, Hong Dai, | † Serena Bagnasco,§ Robert A. Montgomery, and Minnie M. Sarwal

Departments of *Medicine, §Pathology, and |Surgery, Johns Hopkins University, Baltimore, Maryland; †Department of Surgery, University of California, San Francisco, California; and ‡Transplantation Rénale Adulte, Hôpital Necker, Paris, France

ABSTRACT The initial contact point between a recipient’s immune system and a transplanted graft is the vascular endothelium. Clinical studies suggest a pathogenic role for non-HLA antiendothelial cell antibodies (AECAs) in allograft rejection; however, evidence linking AECAs of known specificity to in vivo vascular injury is lacking. Here, we used high-density protein arrays to identify target antigens for AECAs isolated from the sera of recipients of kidney transplants experiencing antibody-mediated rejection in the absence of donor-specific HLA antibodies. Four antigenic targets expressed on endothelial cells were identified: endoglin, Fms-like tyrosine kinase-3 ligand, EGF-like repeats and discoidin I-like domains 3, and intercellular adhesion molecule 4; the first three have been implicated in endothelial cell activation and leukocyte extravasation. To validate these findings, ELISAs were constructed, and sera from an additional 150 renal recipients were tested. All four AECAs were detected in 24% of pretransplant sera, and they were associated with post-transplant donor- specific HLA antibodies, antibody-mediated rejection, and early transplant glomerulopathy. AECA stimulation of endothelial cell cultures increased adhesion molecule expression and production of inflammatory cytokines: regulated on activation, normal T cell expressed and secreted PDGF and RESISTIN. These correlations between in vitro experiments and in vivo histopathology suggest that AECAs activate the vascular endothelium, amplifying the alloimmune response and increasing microvascular damage. Given the growing number of transplant candidates, a better understanding of the antigenic targets, beyond HLA, and mechanisms of immune injury will be essential for improving long-term allograft survival.

J Am Soc Nephrol 26: 1161–1171, 2015. doi: 10.1681/ASN.2013121277

Improvementsintheabilitytodetectalloantibodies have been associated with renal allograft rejection in- in the sera of recipients both before and after renal clude agrin, vimentin, perlecan, Ka-tubulin, protein transplantation and the development of methods to kinase Cz, major histocompatibility complex class I- identify antibody-mediated damage have highlighted related chain A, and angiotensin II type 1 receptor.9 the role of antibodies in both acute and chronic In addition, studies combining proteomics and allograft rejection.1–4 However, the majority of these studies have focused on HLA-speciﬁc antibodies Received December 6, 2013. Accepted August 19, 2014. and their potential to activate complement. The clinical relevance and mechanisms for how alloanti- Published online ahead of print. Publication date available at bodies can contribute to allograft rejection in the www.jasn.org. absence of complement activation, such as in the Correspondence: Dr. Annette M. Jackson, Immunogenetics case of low-level HLA antibodies, are currently un- Laboratory, Johns Hopkins University, 2041 East Monument Street, Baltimore, MD 21215, or Dr. Minnie M. Sarwal, University 5,6 der investigation. of California San Francisco, Division of Transplant Surgery, 505 Reports of allograft rejection in HLA identical Parnassus Avenue, M893, Box 0780, San Francisco, CA 94143. sibling transplants suggest a role for non-HLA anti- Email: [email protected] or [email protected] bodies in some rejections.7,8 Non-HLA antigens that Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 26: 1161–1171, 2015 ISSN : 1046-6673/2605-1161 1161 BASIC RESEARCH www.jasn.org genomics are uncovering tissue-specificnon-HLAantigens capable of initiating humoral responses in recipients of renal transplants.10 Non-HLA antigens expressed on endothelial cells are of particular interest given that the vascularendothelium servesas the point of contact between the recipient’s immune system and the transplanted allograft. A prospective, multicenter clinical trial using peripheral blood endothelial cell precursors (ECPs) as targets showed that nonsensitized recipients transplanted across a positive endothelial cell crossmatch experienced increased rejection and higher serum creatinine values early post-transplantation.11 Most of these rejections were C4d-negative and classified as cellular rejections. Subse- quently, we reported that these antiendothelial cell antibodies (AECAs) are enriched for IgG2 and IgG4, which are IgG sub- classes that activate complement poorly or not at all.12,13 In contrast, identification of AECAs in recipients with broad Figure 1. Antibody mediated injury observed in the AECA positive HLA sensitization or in those undergoing HLA-incompatible Discovery Cohort. Shown are renal biopsies with positive histologic transplantation was associated with an increased incidence scores.1 acquired during the 1.5 years post-transplantation ac- and severity of antibody-mediated rejection (AMR). cording to protocol or at time of dysfunction. Histologic scoring (0–3) – 27–30 Although the endothelial cell crossmatch has been shown to be was performed using updated Banff 1997 2007 criteria. Shown fl clinically useful, it has many technical problems that include the are grades for glomerulitis (g), interstitial (i) and tubular (t) in am- mation, vasculitis (v), and peritubular capillaritis (ptc). C4d staining expression of HLAs on ECPs, which limits our ability to test for was performed on frozen tissue by indirect immunofluorescence. AECAs in HLA-incompatible recipients and impedes our ability to Transplant glomerulopathy (cg) was defined as duplication of the identify those proceeding to transplant across both barriers. glomerular basement membrane as observed on electron and light fi Identi cation of the antigenic targets expressed on ECPs would microscopy. Low-level DR52 HLA-DSA (median fluorescent in- provide the potential to test for AECAs in solid-phase immuno- tensity,1000) was detected in one recipient at the time of biopsy. assays, which in turn, may help in pretransplant risk assessment and provide an opportunity for therapeutic intervention. Here, we describe a proteomics approach to identify novel antigenic targets recipients against approximately 9500 human proteins. Four for AECAs and the development of an ELISA platform to enable proteins expressed on vascular endothelium, endoglin, EGF- testing for these AECAs in patients broadly sensitized to HLA. We like repeats and discoidin I-like domains 3 (EDIL3), intercel- show the effect of these AECAs on endothelial cell cultures in vitro lular adhesion molecule 4 (ICAM4), and Fms-like tyrosine and correlate that with increased in vivo microvascular injury in kinase-3 (FLT3) ligand, were identified in all eluates. Signal patients who test positive for AECAs. intensitiesforthesefourantibodiesweresignificant (endoglin, EDIL3, and FLT3: P,0.001; ICAM4: P,0.03) compared with low-abundance antibodies with signals,2000 relative fluores- RESULTS cent units (Figure 2A, Supplemental Table 2). We also performed analyses of AECA eluates derived from two recipients Identification of Novel Antigenic Endothelial Cell undergoing therapeutic plasmapheresis for rejection. These Targets Using Protein Arrays AECA eluates were derived from endothelial cell crossmatch- AECAs were isolated from a Discovery Cohort of 10 renal positive sera drawn before transplantation, crossmatch-negative transplant recipients whose demographics are provided in sera obtained at the end of treatment, and sera drawn later post- Supplemental Table 1. Most patients (9 of 10) were sensitized treatment (3 and 9 months later) that again tested positive in an to HLA, and all tested positive for AECAs in pretransplant endothelial cell crossmatch. Protein array data, normalized to endothelial cell crossmatch tests. Nine patients experienced total IgG, showed that AECAs for all four protein targets (endoglin, allograft dysfunction and biopsy-proven rejection with noted FLT3 ligand, EDIL3, and ICAM4) decreased after plasmaphe- glomerulitis and peritubular capillaritis (Figure 1). Only one resis and rebounded again in sera that had AECAs detectable recipient had low-level antibody, detected by bead assays only, in crossmatch tests (Figure 2B). to donor HLA (DR52) at the time of rejection. To focus our analyses on AECA target antigens, antibody Expression of Target Antigens on Endothelial Cells eluates were generated using ECPs derived from blood. In brief, Cell phenotype analysis using flow cytometry confirmed each serum was incubated with ECPs, and after wash steps, the surface expression of endoglin, EDIL3, ICAM4, and FLT3 bound antibodies were eluted. Using a high-density protein (receptor for FLT3 ligand) on blood-derived ECPs compared platform, we profiled AECA eluates from 10 Discovery Cohort with an isotype control antibody (data not shown). Analysis of

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was enriched for recipients sensitized to HLA, with 91% (137 of 150) of recipients testing positive for HLA-specific class I and/or II antibodies (Supplemental Table 1). We analyzed the most strongly reacting sera in each ELISA with a signal intensity equal to or greater than the trimmed mean. Fifty- six (37%) sera reacted positively with one or more antigenic targets. Within this group, 36 (24%) sera showed strong reactivity with all four antigen targets (Table 1). Pairwise comparisons performed using the top 36 reacting sera yielded highly significant (P,0.001) correlations in antibody production for these four antigens: endoglin and EDIL3 (R2=0.91, r=0.954), endoglin and ICAM4 (R2=0.89, r=0.94), EDIL3 and ICAM4 (R2=0.87, r=0.93), FLT3 and endoglin (R2=0.74, r=0.86), FLT3 and ICAM4 (R2=0.76, r=0.87), and FLT3 and EDIL3 (R2=0.72, r=0.85). AECA levels specific for these four targets decreased in post-transplant sera (#3 months) in nearly all patients (93%; 140 of 150 patients).

AECA Activation of Endothelial Cell Cultures Primary endothelial cell cultures were established and stimulated with negative control serum, AECA-positive sera, AECA eluates, TNF-a, or a serum containing HLA antibodies. AECA eluates from 5 of the original 10 Discovery Cohort recipients were used as stimulants. The eluates contained a concentra- Figure 2. Identification of antigenic targets for AECAs and the effect tion of AECAs compared with the original serum. of desensitization treatment. (A) Protein array analysis of 14 AECA Cultured endothelial cells were stimulated for 24 hours, eluates derived from endothelial cell crossmatch-positive sera. Relative after which time surface phenotype analysis was performed fluorescence units (RFUs) for antibodies specific for EDIL3, endoglin using flow cytometry to assess endothelial cell activation. We (ENG), ICAM4, and FLT3 Ligand (FLT3LG) were significant compared observed significant increases in the expression of HLA class I with the mean signal intensity of low-abundance antibodies. We (P=0.04) and adhesion molecules E selectin (P=0.02) and defined a positive threshold of 2000 RFU as a cutoff for abundance. ICAM1 (P=0.005) after stimulation with the AECA eluates The error bars represent SEM. (B) Effect of plasmapheresis/intravenous compared with negative control serum (data not shown) or fi Ig (PP) on AECA levels. AECAs speci c for ENG, FLT3LG, EDIL3, and the original AECA-positive sera from which the eluates were ICAM4 decreased after PP treatments and rebounded in a post- derived (Figure 4). The increase in PECAM1 after AECA elu- transplant serum that yielded a positive endothelial cell crossmatch ate stimulation was not statistically different from the negative test. Data shown are from a single renal recipient, and values were P normalized to total IgG. EC, endothelial cell; Txn, transplantation. controls ( =0.12). Stimulation of endothelial cell cultures using TNF-a or serum containing HLA antibodies increased expression of all markers (HLA class I, PECAM1, E selectin, two endothelial cell lines, derived from iliac artery and human and ICAM1) compared with negative controls. umbilical vein, yielded positive staining for endoglin but We assessed the production of inflammatory cytokines and negative staining for EDIL3, ICAM4, and FLT3, even after chemokines after AECA eluate stimulation using a multiplexed TNF-a stimulation (data not shown). immunoassay run on a Luminex platform. Primary endothelial To investigate expression of these antigenic targets in renal cellcultures were stimulated with culture medium alone, negative tissue, immunohistochemistry was performed on rejection bi- control serum, AECA eluates, TNF-a, or serum containing HLA opsies obtained from nine Discovery Cohort recipients. Figure 3 antibodies specificfortheECPdonor.After72hours,culture illustrates representative staining for endoglin and FLT3, which supernatants were harvested and tested in an Affymetrix Procarta were expressed on arterial endothelium and glomerular and peri- human immunoassay; 44 of 54 analytes were negative in all su- tubular capillaries. Concomitant staining of biopsy tissue for FLT3 pernatants tested. IL-8, IL-1 receptor antagonist, and Serpin E1 ligand, EDIL3, and ICAM4 yielded negative results. were detected in all test cultures, including those without stimulation (culture medium alone). Inflammatory cytokines PDGF, Incidence of AECAs Using Antigen-Specific ELISAs RANTES (also known as CCL5), and RESISTIN were increased Sera from 150 sequential recipients of renal transplants for in cultures stimulated with the AECA eluates compared with whom there were adequate pre- and post-transplant negative controls or cultures stimulated with TNF-a or HLA (#3 months) samples were tested using MSD ELISAs specific antibodies (Figure 5). In contrast, chemokines CCL3, CCL4, for endoglin, EDIL3, ICAM4, and FLT3. This retrospective CXCL5,andCXCLseemedtobedecreasedinculturesstimulated study cohort was similar to the Discovery Cohort in that it with the AECA eluates compared with other stimulants.

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showed less glomerulitis but more peritubular capllaritis, C4d positivity, and chronic vascular changes than the negative group. The incidence of HLA-DSA at time of biopsy was high, reflecting the high degree of HLA sensitization and the inclusion of incompatible transplants in these patient cohorts (Table 1). HLA-DSA was detected atthetimeofbiopsyin76%ofbiopsies from the ELISA-positive recipients, 45% of biopsies from the intermediate positive recipients, and 47% of biopsies from ELISA-negative recipients. Recipients who tested strongly positive for AECAs had a greater number of DSAs at time of biopsy and an increased incidence of antibodies to both HLA classes I and II antigens. From a subsequent analysis, we eliminated biopsies with high-level HLA-DSA sufficient to yield a positive flow cytometric crossmatch (FCXM) or cytotoxicity lymphocyte crossmatch test Figure 3. Expression of endoglin and FLT3 on renal endothelium. Immunohistochemistry to examine the effect of low-level HLA-DSA performed on biopsies taken at time of rejection shows expression of (A) endoglin and (B) and AECAs. Among remaining biopsies with FLT3 on glomerular and peritubular microvasculature and arteries. Data shown are rep- no or low-level HLA-DSA, which were de- resentative of biopsies tested from nine Discovery Cohort recipients. tected only by bead assays, more microvascular injury was still observed in the strongly positive AECA recipients versus negative re- Clinical Outcomes for Recipients Testing AECA- cipients (Figure 6, Table 2). Histopathology scores for microvas- Positive cular injury were not significantly increased in biopsies from To bring this study full circle, we performed a preliminary recipients with intermediate levels of AECAs with no or low analysis of the clinical outcomes for 40 recipients with the HLA-DSA. lowest reacting sera on the ELISAs, an intermediate group of 20 Serum creatinine levels in the immediate post-transplant recipients who tested above the trimmed mean for one to three period (7 days) were significantly higher in the ELISA-positive target antigens, and 36 recipients who tested strongly positive group (P=0.004), corresponding to the high incidence of de- for antibodies against all four targets: endoglin, EDIL3, layed graft function. The mean serum creatinine at 1 month ICAM4, and FLT3 (Table 1). Sera from the remaining recipi- was higher in the ELISA-positive group (1.8061.07) com- ents tested below the trimmed mean in ELISAs for all four pared with the ELISA-negative group (1.3861.02), but the target antigens. The demographics for these three ELISA co- difference was not significant (P=0.09). Mean serum creati- horts were similar for all pretransplantation characteristics, nine levels at 3 months, 1 year, and $1.5 years were also higher including HLA-DSA strength at the time of transplantation. the ELISA-positive cohort but not significantly different. In total, 28% of the ELISA-positive group and 25% of the intermediate group experienced delayed graft function, re- quiring post-transplant dialysis, compared with 15% in the DISCUSSION ELISA-negative cohort (Table 2). The incidence of cellular rejection was not significantly different between the three In this study, a proteomic approach was used to identify novel groups; however, AMR was significantly higher in recipients antigenic targets for AECAs associated with renal allograft who tested positive for AECAs (intermediate: 15%, P=0.04; dysfunction. Protein array analysis of AECAs eluted from ECPs strongly positive: 19%, P=0.007) compared with 4% in re- identified endoglin, EDIL3, ICAM4, and FLT3 ligand as target cipients who tested ELISA-negative. Analyses of 86 biopsies antigens. Endoglin is constitutively expressed within the from 36 recipients with the strongest reacting sera (68 indi- vasculature and has been proposed to play a role in endothelial cation and 18 protocol biopsies) revealed significantly more cell activation and inflammation.14 FLT3 is a tyrosine kinase glomerulitis, peritubular capllaritis, C4d positivity, and trans- receptor for the cytokine FLT3 ligand. In mice, Thomson and plant glomerulopathy than biopsies from 40 ELISA-negative re- colleagues15 showed that FLT3 stimulation can increase cipients (36 indication and 34 protocol biopsies) (Figure 6, Table 2). RANTES (CCL5) production by renal endothelial cells, result- The intermediate group (31 indication and 36 protocol biopsies) ing in the recruitment of CD45+ mononuclear cells.

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Table 1. Patient demographics for AECA ELISA categories ELISA Categories Negative (n=40) Intermediate (n=20) P Value Strongly Positive (n=36) P Value ELISA values, mean; median Endoglin 1058; 992 6486; 6357 ,0.001 37,432; 26,101 ,0.001 FLT3 1311; 1105 5499; 5876 ,0.001 29,876; 19,104 ,0.001 EDIL3 324; 0 4437; 4397 ,0.001 34,213; 23,290 ,0.001 ICAM4 3215; 3272 15,378; 13,077 ,0.001 56,839; 32,896 ,0.001 Recipient age (yr), mean6SD 52616 49613 0.47 47615 0.22 Race, % not white 40 40 .0.99 50 0.49 Men, % 25 50 0.08 44 0.09 Previous transplantation, % 40 50 0.58 39 .0.99 HLA sensitization,a %9090.0.99 97 0.36 Mean CPRAb (CDC-XM, FCXM), % 27, 37 27, 38 0.94 35, 44 0.48 Original ABO or HLA barrier,c % ABOi 10 15 0.68 18 0.50 CDC-XM+ 2 0 .0.99 6 0.60 FCXM+ 15 15 .0.99 19 0.76 FCXM2, DSA+ 48 55 0.78 50 .0.99 No DSA 35 30 0.78 25 0.45 Donor, mean age (yr)6SD 38613 43612 0.12 42616 0.25 Live donor, % 45 55 42 Deceased donor, % 55 45 0.59 58 0.82 HLA-A;B;DR;DQ mismatch, mean6SD 4.762.3 4.162.7 0.41 5.261.5 0.21 Plasmapheresis treatments No pre- or post-treatments, % 50 35 0.41 42 0.50 Pretransplant, mean, median 1.0, 0.0 1, 1 0.67 0.5, 0.0 0.17 Post-transplant, mean, median 3.6, 1.0 3, 2 0.86 4.5, 2.0 0.54 Anti-CD25 induction, % 17 20 .0.99 15 0.76 Thymoglobulin induction, % 83 80 .0.99 85 0.76 Rituximab induction, % 33 45 0.40 38 0.63 CPRA, calculated panel reactive antibody; CDC-XM, cytotoxicity crossmatch; ABO, blood group antigen; ABOi, blood group incompatible; DSA, donor HLA- specific antibody; HLA-A;B;DR;DQ, histocompatibility antigens. aHLA-specific antibody detected on Luminex bead immunoassays. bCPRA was determined for HLA antibodies of sufficient strength to yield a positive CDC-XM or FCXM. cOriginal donor HLA-specific antibody (DSA) strength before desensitization treatment.

EDIL3 was not detected by immunohistochemistry in renal absence of detectable complement activation, which was biopsies taken at the time of rejection from our Discovery observed in our Discovery Cohort, was included.2 Gene array Cohort, but inflammation has been shown to reduce EDIL3 data from Sis et al.19 have shown that increased endothelial cell expression on vascular endothelium.16 In mice, EDIL3 (Del-1) transcripts are a more sensitive biomarker for AMR than de- functions as an inhibitor of LFA1-dependent cell adhesion, tection of C4d in acute renal biopsies. We and others have resulting in reduced leukocyte extravasation from blood into reported hyperacute rejections in patients positive for AECAs underlying tissues. Inflammation acts as a switch to upregulate with no detectable HLA-DSA.13,20 Thus, there is growing ev- expression of activation molecules, such as ICAM-1 and vas- idence that antibody-induced endothelial cell activation, in- cular cell adhesion molecule 1, and downregulate inhibitory dependent of complement, may be sufficient for immune cell molecules, such as EDIL3. Therefore, the inability to detect recruitment and microvascular injury. Three of the AECA tar- EDIL3 at the time of a rejection biopsy may not preclude its gets identified in this study (endoglin, EDIL3, and FLT3) have presence on endothelial cells during the initial stages of rejec- been implicated in endothelial activation and leukocyte ex- tion. The final protein identified in the AECA eluates was travasation, and these findings are consistent with our in vitro ICAM4 (previously named the Landsteiner–Wiener blood data showing that AECA stimulation of endothelial cells re- group). This molecule is part of the ICAM family of adhesion sults in the upregulation of adhesion and HLA molecules and molecules and believed to be red blood cell lineage-specific.17 the production of inflammatory chemokines and cytokines. Expression of ICAM4 on renal endothelium could not be val- Admittedly, the histopathology analysis was confounded by idated using immunohistochemistry; however, vascular the presence of HLA-DSA in many recipients at time of re- expression has been documented in microarray literature.18 jection, thus weakening our ability to prove a causal link for In the 2011 Banff Conference Allograft Pathology Report, AECAs. However, the AECA ELISA-positive and -negative co- the heterologous phenotypes associated with AMR in renal horts used for outcome analyses were matched for all relative transplantation were discussed and recognition of AMR in the criteria at time of transplantation, including breadth of

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recipients who tested positive in endothelial cell crossmatch tests and experienced allograft rejection in the ﬁrst 3 months post-transplant in the absence of donor HLA-speciﬁc antibody were tested on a protein array platform. ELISA tests were performed on 150 sequential recipients of renal transplants for whom there were adequate pre- and post-transplant (#3 months) sera. Patient demographics are provided in Sup- plemental Table 1.

Immunosuppression Maintenance immunosuppression included mycophenolatemofetil(2g/d),tacrolimus(serum level of 8–10 ng/ml), and prednisone (30 mg/d). Prednisone was reduced to 20 mg/d when tacrolimus reached therapeutic range (8–12 ng/dl) and ta- pered thereafter to 5 or 10 mg/d. Intraoperative induction therapy was either anti–IL-2 receptor antibody (anti-CD25, daclizumab at 2 mg/kg) or Figure 4. Endothelial cell cultures stimulated with AECA eluates upregulate markers of Thymoglobulin (1.5 mg/kg per day for 5 days). activation. Primary endothelial cell cultures werestimulatedwithAECA-positivesera,AECA eluates, TNF-a (10 ng/well), or an HLA antibody-positive serum. Cell surface phenotype HLA and ABO incompatible pairs were included analysis was performed 24 hours poststimulation using flow cytometry. Comparisons of in this study. Desensitization included al- median fluorescence values were made between cells stimulated with AECA eluates versus ternate day plasmapheresis immediately fol- recipient sera (P values shown). Cumulative data were obtained from seven independent lowed by low-dose (100 mg/kg) intravenous Ig experiments, and AECA eluates from five Discovery Cohort recipients were tested. Aby, (Cytogam-CSL Behring, King of Prussia, PA). antibody; PECAM1, platelet/endothelial cell adhesion molecule 1. Mycophenolate mofetil (2 g/d) and tacrolimus (serum level of 8–10 ng/ml) were initiated with the start of plasmapheresis treatments. The num- sensitization and strength of HLA-DSA at final crossmatch. ber of treatments was dependent on ABO or donor HLA-specificanti- The divergence of these groups post-transplantation with re- body (HLA-DSA) titer. Biopsy-confirmed acute cellular rejection was gards to the severity of rejection and the presence of HLA-DSA treated with pulse solumedrol at cumulative doses of 30–50 mg/kg in at time of rejection supports our hypothesis that pretransplant three to six doses and increased baseline target levels for tacrolimus AECA detection in recipients sensitized to HLA identifies and/or mycophenolate mofetil. Biopsy-confirmed acute AMR was those at higher risk for allograft rejection. Given the increased treated with plasmapheresis and/or rituximab and/or intravenous Ig incidence of transplant glomerulopathy in the AECA-positive and pulse solumedrol. cohort, additional studies evaluating the effect of AECAs in long-term allograft survival are needed. HLA Antibody Detection and Crossmatch Tests RoutinetestingforAECAshasnotbeenrealized,inpart,because HLA-specific antibodies were evaluated before transplantation and at of a paucity of tools for inexpensive high-throughput screening of time of biopsy using solid-phase immunoassays (Lifecodes classes I patient sera. The use of cell-based crossmatch methods has been and II ID panels; Immucor-Lifecodes, Stamford, CT; Single Antigen problematic because of the inefficiency of testing single recipient/ Beads; One Lambda, Canoga Park, CA) performed on a Luminex donor pairs and the inability to identify non-HLA antibodies in platform. Crossmatch tests with donor T and B cells were performed patients with known HLA sensitization. In this study, we developed using standard cytotoxicity and/or FCXM protocols. Calculated panel an ELISA platform to increase the sensitivity and efficiency of reactive antibody was determined for antibodies sufficient to yield a AECA detection and provided mechanistic data to investigate the positive cytotoxicity or FCXM using virtual crossmatch methods and role of AECAs in microvascular injury. the online UNOS CPRA calculator.21 Non-HLA AECAs were detected using an FCXM performed using angiopoietin receptor positive (Tie-2+) peripheral blood ECPs22 (XM-ONE; Absorber AB, CONCISE METHODS Stockholm, Sweden) acquired on a BD FACSAria using FACSDiva (version 6.1.1; BD Biosciences, Franklin Lakes, NJ). Study Patients Clinical data and stored sera from 160 recipients of renal transplants Non-HLA AECA Eluates transplanted between 2009 and 2011 were studied retrospectively after AECA eluates were derived from adsorbing antibody from endothelial institutional review board approval. Sera from a discovery group of 10 cell crossmatch-positive sera onto Tie-2+ ECPs isolated from

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contained approximately 9500 recombinant human proteins expressed as N-terminal GST fusion proteins and spotted on nitrocellulose- coated glass slides. Established protocols (http:// www.invitrogen.com) were followed24,25 for sample preparation, blocking, probing, drying by centrifugation, scanning, and data acquisi- tion. Slides were scanned using an Axon GenePix 4000B Scanner (Molecular Devices, Sunnyvale, CA) and GenePix pro 6.0 software (Molecular Devices). We used the data analysis software Pro- toArray Prospector 5.2 to analyze any bound IgG antibody detected by the secondary Alexa Fluor 647-conjugated antibody. Binding of the secondary antibody on the microarray was then quan- tified by measuring the fluorescence intensity of each feature on the slide. Data generation and normalization involved calculating appropriate fluorescent signal values by taking into account corrections for background and negative control features printed on the microarray. Z factors for all of the corrected intensities of the human protein features were calculated, and the features that had signal intensities greater than the cutoff value of 2000 were considered as abundant signal. Pathway analysis was performed using Inge- Figure 5. Endothelial cell cultures stimulated with AECA eluates differentially affect nuity Pathway analysis (Ingenuity Systems; fl production of in ammatory cytokines and chemokines. Primary endothelial cell cultures www.ingenuity.com) and the GO-Elite Pathway were stimulated with culture media alone, AECA eluates, an HLA antibody-positive serum, Analysis Tool (http://www.genmapp.org/go_elite/ or TNF-a (10 ng/well). Culture supernatant was tested 72 hours poststimulation using go_elite.html). a Procarta human 54 analyte immunoassay acquired on a Luminex xMAP multiplex platform. Median fluorescent intensities (MFIs) for differentially expressed analytes and positive control standards are shown. Data are representative of four independent ex- Endothelial Cell Antigen-Specific periments, and testing AECA eluates were derived from five Discovery Cohort recipients. ELISAs Production of inflammatory cytokines PDGF, regulated on activation normal T cell ex- Endothelial cell-specific antigen targets identi- pressed and presumably secreted (RANTES; also known as CCL5), and RESISTIN were fied by protein arrays were validated using the increased after stimulation with the AECA eluates compared with negative controls MSD ELISA platform (Meso Scale Discovery, , , , a (P 0.001, P 0.001, and P 0.001, respectively) or cells stimulated with TNF- or HLA Gaithersburg, MD). Recombinant proteins/an- , , antibodies (P 0.001, P 0.001, and P=0.002, respectively). In contrast, chemokines tigens obtained from Abcam, Inc. (Cambridge, CCL3, CCL4, CXCL5, and CXCL decreased in cultures stimulated with the AECA eluates MA) included EDIL3 (catalog no. ab94549), compared with stimulation with TNF-a or HLA antibodies (P,0.001, P=0.05, P=0.07, and FLT3 (catalog no. 83996), and endoglin (catalog P=0.04, respectively). Pos, positive; Std, standard. no. ab95043), and recombinant ICAM4 (catalog no. H00003386-P) was purchased from Abnova surrogate donors for whom there was no HLA-DSA. The serum-to- (Walnut, CA). To increase detection specificity and sensitivity, each cell ratio, incubation, and wash steps were consistent with the antigen was biotin-conjugated and incubated with diluted serum before endothelial cell crossmatch procedure. Antibody was acid-eluted 1:10 coating the antigen–antibody complex onto the avidin-coated ELISA of the original serumvolume as previously described23 and dialyzed 18 plate. Biotin labeling of each antigen (5 mg) was performed using hours against PBS. AECA eluates were retested in endothelial cell EZ-Link Sulfo-NHS-LC_Biotin (catalog no. PI21335), the EZ BIOTIN crossmatch tests to ensure antibody integrity and specificity. QUANTIFICATION KIT (catalog no. PI28005), and Zeba* Spin De- salting Columns (7K MWCO, 5 ml; catalog no. PI89892) following the Protein Microarray Analyses manufacturer’s protocol. The serum was diluted 1:75 with 2% Blocker A ProtoArray Human Protein Microarrays v5.0 (Life Technologies, Foster (catalog no. R93AA-1; Meso Scale Discovery) in TPBS after titration City, CA) were used to profile antibodies present in 14 AECA eluates from studies showed it to be the optimal dilution. Equal parts of diluted 10 recipients who were endothelial cell crossmatch-positive. For two serum (50 ml) and biotin-labeled antigen (5 ng) or 2% Blocker A in recipients, analysis was performed on AECA eluates derived from sera TPBS were added to a Streptavidin Gold Plate (catalog no. L15SA-1; obtained before and after desensitization treatments. The protein arrays Meso Scale Discovery) and incubated for 90 min at room temperature

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Table 2. Correlation between AECA ELISAs and clinical outcome ELISA Categories Negative (n=40) Intermediate (n=20) P Value Strongly Positive (n=36) P Value Graft losses,a no. 0 0 .0.99 1 0.47 Delayed graft function,b % 15 25 0.48 28 0.26 Acute rejectionc,1 yr, % biopsies 25 32 0.76 37 0.33 CMR, N (%) 15(21) 13(19) 0.83 16(19) 0.69 AMR, N (%) 3 (4) 10 (15) 0.04 16 (19) ,0.01 Time of first rejection (d), mean, median 131, 102 76, 17 0.63 67, 19 0.18 Donor-specific HLA antibody (HLA-DSA) All biopsies, % 44 34 0.16 75 ,0.001 No/low HLA-DSA biopsies, % 25 24 0.39 38 ,0.01 Number DSA all biopsies, mean6SD 0.861.1 1.361.5 0.04 2.561.9 ,0.001 Class I, N (% of biopsies) 11 (14) 5 (7) 0.18 14 (17) .0.99 Class II, N (% of biopsies) 12 (14) 14 (21) 0.66 24 (28) 0.13 Classes I and II, N (% of biopsies) 8 (11) 5 (7) 0.56 26 (31) ,0.01 Glomerulitis (0–3), mean6SD All biopsies 0.460.5d 0.260.4e 0.004 0.760.9f 0.004 No/low HLA-DSA 0.460.5 0.160.3 ,0.001 0.560.6 0.47 Inflammation (0–3), mean6SD All biopsies 0.860.9 0.860.9 0.79 0.760.9 0.79 No/low HLA-DSA 0.760.9 0.560.6 0.14 0.660.7 0.53 Tubulitis (0–3), mean6SD All biopsies 0.760.8 0.860.9 0.78 0.860.8 0.41 No/low HLA-DSA 0.660.8 0.560.6 0.43 0.860.8 0.11 Vasculitis (0–3), mean6SD All biopsies 0.160.2 0.260.5 0.18 0.360.6 0.004 No/low HLA-DSA 0.160.2 0.060.2 0.82 0.160.4 0.23 Peritubular capllaritis (0–3), mean6SD All biopsies 0.661.1 1.161.3 0.04 1.461.4 ,0.001 No/low HLA-DSA 0.460.9 0.661.1 0.33 1.161.3 0.002 C4d (0–3), mean6SD All biopsies 0.160.4 0.661.1 0.004 0.761.2 ,0.001 No/low HLA-DSA 0.060.0 0.060.2 0.22 0.360.9 0.02 Transplant glomerulopathy (0–3), mean6SD All biopsies 0.160.3 0.060.3 0.85 0.360.7 0.01 No/low HLA-DSA 0.160.3 0.060.0 0.35 0.360.7 0.05 IFTA (0–3), mean6SD All biopsies 1.461.4 1.961.8 0.12 1.561.5 0.43 No/low HLA-DSA 1.061.3 1.461.5 0.07 1.761.5 ,0.01 Cv (0–3), mean6SD All biopsies 0.760.8 1.161.1 0.02 0.961.0 0.11 No/low HLA-DSA 0.660.8 0.961.0 0.08 0.960.9 0.08 Serum creatinine, mean6SD 7d 2.3962.25 3.2762.52 0.19 4.6964.27 0.004 1 mo 1.3861.02 1.7760.97 0.16 1.8061.07 0.09 3 mo 1.3360.64 1.9061.62 0.16 1.6361.40 0.28 12 mo 1.4060.56 1.7361.47 0.40 1.6161.47 0.48 Last available value 1.73 61.47 2.0562.10 0.55 2.5962.88 0.11 Post-transplant, mo 18.8613.4 1962.1 0.99 24.5614.9 0.09 CMR, cellular-mediated rejection; DSA, donor HLA-specific antibody; IFTA, interstitial fibrosis and tubular atrophy; Cv, chronic vascular changes. aGraft loss because of accelerated vascular rejection. bPost-transplant dialysis. cAMR or CMR using updated Banff 1997–2007 criteria.27–30 dIn total, 70 biopsies (34 protocol) and 56 biopsies (28 protocol) with no or low HLA-DSA (detected by Luminex only). eIn total, 67 biopsies (36 protocol) and 46 biopsies (30 protocol) with no or low HLA-DSA (detected by Luminex only). fIn total, 86 biopsies (18 protocol) and 55 biopsies (16 protocol) with no or low HLA-DSA (detected by Luminex only).

1168 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 1161–1171, 2015 www.jasn.org BASIC RESEARCH

Figure 6. Histologic scores for recipients who were AECA ELISA strongly positive (n=28) and negative (n=24). Renal biopsies with positive histologic scores.1 were acquired during the first year post-transplantation according to protocol or at the time of dysfunction. Shown are scores from all biopsies or a subset of biopsies when HLA-DSA was not detected at time of biopsy or detected at a negative FCXM level.21 Histologic scoring (0–3) was performed using updated Banff 1997–2007 criteria.27–30 Shown are grades for glomerulitis (g), interstitial (i) and tubular (t) inflammation, vasculitis (v), and peritubular capillaritis (ptc). C4d staining was performed on frozen tissue by indirect immunofluorescence. Transplant glomerulopathy (cg) was defined by duplication of the glomerular basement membrane as observed on electron and light microscopy. Additional details and statistics are provided in Table 2. with shaking (300 rpm). The plate was blocked with 5% Blocker A match test with sera from the Discovery Cohort as previously (150 ml) for 90 minutes at room temperature with shaking and washed described with minor modifications.26 In brief, ECPs were isolated one time with 300 ml TPBS, and 100 ml diluted goat anti-human using Tie-2 magnetic beads (XM-ONE Absorber AB), plated at a SULFO-TAG (1:1000) detection antibody was added and incubated in 1000-cells/mm2 density in fibronectin (1 mg/well)-coated wells, the dark for 60 minutes at room temperature with shaking. After three and cultured for 2–3 weeks in supportive media (EGM-2 Bullet Kit; washes with 300 ml TPBS, 150 ml reading buffer was added to each well, Lonza, Walkersville, MD) to allow for maturation. Endothelial cell and the plate was immediately read with an SECTOR Imager 2400 lines (CRL-2606; ATCC; HuVEC, gift of Mark K. Halushka) were (Meso Scale Discovery). Reactivity above the trimmed mean was con- cultured as above; cultures were passed when 70%–80% confluent sidered positive, and changes between pre- and post-transplant anti- and analyzed within the first five passages. body levels were determined by increases or decreases that exceeded a 95% confidence interval for each ELISA. Analyses after Endothelial Cell Culture Stimulation Primary endothelial cell cultures were washed two times with wash buffer Endothelial Cell Culture (13 PBS with 5% heat-inactivated FCS). Cells were stimulated using Primary endothelial cell cultures were established using Tie-2+ ECPs 100 ml negative control AB serum, endothelial cell crossmatch positive isolated from donors who yielded a positive endothelial cell cross- sera, AECA eluates, TNF-a (10 ng/well; Abcam, Inc.), or a serum

J Am Soc Nephrol 26: 1161–1171, 2015 Endothelial Cell Reactive Antibody 1169 BASIC RESEARCH www.jasn.org containing antibodies specific for HLA antigens of the ECP donor. This work was supported by a grant from the National Kidney Cells were incubated for 1 hour, after which time 1 ml supportive Foundation of Maryland (to A.M.J.). media was added to each well. Cell surface phenotype analysis was performed 24 hours poststimulation. Cells were removed with 0.25% trypsin, washed, and stained according to standard protocols. DISCLOSURES mAbs included peridinin chlorophyll protein complex-conjugated None. CD54 (clone HA58), phycoerythrin-conjugated CD62E (clone 68–5H11), fluorescein isothiocyanate-conjugated HLA class I (clone G46–2.6) and CD3 (clone SK7), and Alexa Fluor 647-congugated CD31 (clone WM59; REFERENCES BD Biosciences). Rabbit anti-human polyclonal antibodies included CD105 (endoglin, ab21224), CD242 (ICAM4, ab112554), EDIL3 1. McKenna RM, Takemoto SK, Terasaki PI: Anti-HLA antibodies after – (ab74775), and CD135 (FLT3, ab37847; Abcam, Inc.). Allophycocyanin- solid organ transplantation. Transplantation 69: 319 326, 2000 2. Mengel M, Sis B, Haas M, Colvin RB, Halloran PF, Racusen LC, Solez K, conjugated goat anti-rabbit IgG was purchased from R&D Systems (Min- Cendales L, Demetris AJ, Drachenberg CB, Farver CF, Rodriguez ER, neapolis, MN). Cells were acquired (2000 gated events) and analyzed using Wallace WD, Glotz D; Banff meeting report writing committee: Banff BD FACSAria and FACSDiva (version 6.1.1; BD Biosciences) and/or De 2011 Meeting report: New concepts in antibody-mediated rejection. Novo Software (Los Angeles, CA). After 72 hours of stimulation, culture Am J Transplant 12: 563–570, 2012 supernatants were tested using the Procarta human 54 analyte immu- 3. Sis B, Jhangri GS, Riopel J, Chang J, de Freitas DG, Hidalgo L, Mengel M, Matas A, Halloran PF: A new diagnostic algorithm for antibody- noassay (Affymetrix Inc., Santa Clara, CA) according to the manufac- mediated microcirculation inflammation in kidney transplants. Am J ’ turer s protocol and acquired on a Luminex xMAP multiplex platform. Transplant 12: 1168–1179, 2012 Each assay included a positive control standard for each target protein. 4. Loupy A, Hill GS, Jordan SC: The impact of donor-specificanti-HLAan- tibodies on late kidney allograft failure. Nat Rev Nephrol 8: 348–357, 2012 5. Morrell CN, Murata K, Swaim AM, Mason E, Martin TV, Thompson LE, Histopathogy Ballard M, Fox-Talbot K, Wasowska B, Baldwin WM 3rd: In vivo platelet- All biopsies performed during the first year post-transplantation were endothelial cell interactions in response to major histocompatibility included in the analysis. Biopsies were performed at the time of graft complex alloantibody. 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C4d staining was considered positive if present in Stegall MD, Amer H: Antibody-mediated rejection following trans- $50% of the peritubular capillaries with intensity$1+ (C4d2–3). Ad- plantation from an HLA-identical sibling. Nephrol Dial Transplant 25: 307–310, 2010 ditional staining of biopsy tissue was performed using rabbit anti- 9. Sigdel TK, Sarwal MM: Moving beyond HLA: A review of nHLA anti- human polyclonal antibodies reactive with CD105 (RB9291P1; bodies in organ transplantation. Hum Immunol 74: 1486–1490, 2013 Thermo Fisher Scientific, Waltham, MA), CD242 (ICAM4, 10. Li L, Sigdel T, Vitalone M, Lee SH, Sarwal M: Differential immunoge- ab112554), EDIL3 (ab74775), and CD135 (FLT3, ab37847; Abcam, nicity and clinical relevance of kidney compartment specific antigens – Inc.). Detection was performed using a horseradish peroxidase after renal transplantation. JProteomeRes9: 6715 6721, 2010 11. Breimer ME, Rydberg L, Jackson AM, Lucas DP, Zachary AA, Melancon JK, polymer-conjugated secondary antibody (SuperPicture Polymer De- VonVisger J, Pelletier R, Saidman SL, Williams WW Jr., Holgersson J, tection Kit; Life Technologies, Grand Island, NY). Tyden G, Klintmalm GK, Smith D, Coultrup S, Sumitran-Holgersson S, Grufman P: Multicenter evaluation of a novel endothelial cell crossmatch test in kidney transplantation. Transplantation 87: 549–556, 2009 Statistical Methods 12. Jackson AM, Lucas DP, Melancon JK, Desai NM: Clinical relevance and Protein array data were analyzed by Prospector Analyzer (Life Technol- IgG subclass determination of non-HLA antibodies identified using ogies) using robust linear model normalization.31 A minimum relative endothelial cell precursors isolated from donor blood. Transplantation fluorescent unit.500 and a Z factor of 0.4 were required for positive 92: 54–60, 2011 13. Jackson AM, Kuperman MB, Montgomery RA: Multiple hyperacute detection. Summary statistics, including mean, trimmed mean, median, rejections in the absence of detectable complement activation in a fi fi fi con dence norm distribution, correlation coef cient, coef cient of dif- patient with endothelial cell reactive antibody. Am J Transplant 12: ference, and SD, were calculated using Microsoft Excel. Statistical sig- 1643–1649, 2012 nificance was determined using chi-squared and t tests (two tailed), and 14. Docherty NG, López-Novoa JM, Arevalo M, Düwel A, Rodriguez-Peña A, P values,0.05 were considered significant. Pérez-Barriocanal F, Bernabeu C, Eleno N: Endoglin regulates renal ischaemia-reperfusion injury. Nephrol Dial Transplant 21: 2106–2119, 2006 15. Coates PT, Colvin BL, Ranganathan A, Duncan FJ, Lan YY, Shufesky WJ, ACKNOWLEDGMENTS Zahorchak AF, Morelli AE, Thomson AW: CCR and CC chemokine expression in relation to Flt3 ligand-induced renal dendritic cell mobili- zation. Kidney Int 66: 1907–1917, 2004 We thank Drs. Andrea A. Zachary and Mary S. Leffell for critical 16. Choi EY, Chavakis E, Czabanka MA, Langer HF, Fraemohs L, reading of this manuscript. Economopoulou M, Kundu RK, Orlandi A, Zheng YY, Prieto DA, Ballantyne

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CM,ConstantSL,AirdWC,PapayannopoulouT,GahmbergCG,UdeyMC, LW, Gibson IW, Glotz D, Goldberg JC, Grande J, Halloran PF, Hansen Vajkoczy P, Quertermous T, Dimmeler S, Weber C, Chavakis T: Del-1, an HE, Hartley B, Hayry PJ, Hill CM, Hoffman EO, Hunsicker LG, Lindblad endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory AS, Yamaguchi Y: The Banff 97 working classification of renal allograft cell recruitment. Science 322: 1101–1104, 2008 pathology. Kidney Int 55: 713–723, 1999 17. Toivanen A, Ihanus E, Mattila M, Lutz HU, Gahmberg CG: Importance of 28. Solez K, Colvin RB, Racusen LC, Sis B, Halloran PF, Birk PE, Campbell molecular studies on major blood groups—intercellular adhesion PM, Cascalho M, Collins AB, Demetris AJ, Drachenberg CB, Gibson IW, molecule-4, a blood group antigen involved in multiple cellular inter- Grimm PC, Haas M, Lerut E, Liapis H, Mannon RB, Marcus PB, Mengel actions. Biochim Biophys Acta 1780: 456–466, 2008 M, Mihatsch MJ, Nankivell BJ, Nickeleit V, Papadimitriou JC, Platt JL, 18. Su AI, Cooke MP, Ching KA, Hakak Y, Walker JR, Wiltshire T, Orth AP, Randhawa P, Roberts I, Salinas-Madriga L, Salomon DR, Seron D, Sheaff Vega RG, Sapinoso LM, Moqrich A, Patapoutian A, Hampton GM, M, Weening JJ: Banff ‘05 Meeting Report: Differential diagnosis of Schultz PG, Hogenesch JB: Large-scale analysis of the human and chronic allograft injury and elimination of chronic allograft nephropathy mouse transcriptomes. Proc Natl Acad Sci U S A 99: 4465–4470, 2002 (‘CAN’). Am J Transplant 7: 518–526, 2007 19. Sis B, Jhangri GS, Bunnag S, Allanach K, Kaplan B, Halloran PF: En- 29. Solez K, Colvin RB, Racusen LC, Haas M, Sis B, Mengel M, Halloran PF, dothelial gene expression in kidney transplants with alloantibody in- Baldwin W, Banfi G, Collins AB, Cosio F, David DS, Drachenberg C, dicates antibody-mediated damage despite lack of C4d staining. Am J Einecke G, Fogo AB, Gibson IW, Glotz D, Iskandar SS, Kraus E, Lerut E, Transplant 9: 2312–2323, 2009 Mannon RB, Mihatsch M, Nankivell BJ, Nickeleit V, Papadimitriou JC, 20. Sumitran-Karuppan S, Tyden G, Reinholt F, Berg U, Moller E: Hyper- Randhawa P, Regele H, Renaudin K, Roberts I, Seron D, Smith RN, acute rejections of two consecutive renal allografts and early loss of the Valente M: Banff 07 classification of renal allograft pathology: Updates third transplant caused by non-HLA antibodies specificforendothelial and future directions. Am J Transplant 8: 753–760, 2008 cells. Transpl Immunol 5: 321–327, 1997 30. Racusen LC, Colvin RB, Solez K, Mihatsch MJ, Halloran PF, Campbell 21. Zachary AA, Sholander JT, Houp JA, Leffell MS: Using real data for a PM, Cecka MJ, Cosyns JP, Demetris AJ, Fishbein MC, Fogo A, virtual crossmatch. Hum Immunol 70: 574–579, 2009 Furness P, Gibson IW, Glotz D, Hayry P, Hunsickern L, Kashgarian M, 22. Jackson AM, Lucas DP, Badders JL: A flow cytometric crossmatch test Kerman R, Magil AJ, Montgomery R, Morozumi K, Nickeleit V, using endothelial precursor cells isolated from peripheral blood. Randhawa P, Regele H, Seron D, Seshan S, Sund S, Trpkov K: Anti- Methods Mol Biol 1034: 319–329, 2013 body-mediated rejection criteria - an addition to the Banff 97 clas- 23. Lucchiari N, Panajotopoulos N, Xu C, Rodrigues H, Ianhez LE, Kalil J, sification of renal allograft rejection. Am J Transplant 3: 708–714, Glotz D: Antibodies eluted from acutely rejected renal allografts bind to 2003 and activate human endothelial cells. Hum Immunol 61: 518–527, 2000 31. Sboner A, Karpikov A, Chen G, Smith M, Mattoon D, Freeman-Cook L, 24. Sigdel TK, Li L, Tran TQ, Khatri P, Naesens M, Sansanwal P, Dai H, Hsieh SC, Schweitzer B, Gerstein MB: Robust-linear-model normalization to re- Sarwal MM: Non-HLA antibodies to immunogenic epitopes predict the evo- duce technical variability in functional protein microarrays. JProteome lution of chronic renal allograft injury. JAmSocNephrol23: 750–763, 2012 Res 8: 5451–5464, 2009 25. Sigdel TK, Shoemaker LD, Chen R, Li L, Butte AJ, Sarwal MM, Steinberg GK:Immuneresponseprofiling identifies autoantibodies specificto Moyamoya patients. Orphanet J Rare Dis 8: 45, 2013 26. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, See related editorial, “Renal Allograft Rejection: Pieces of the Puzzle,” on Witzenbichler B, Schatteman G, Isner JM: Isolation of putative pro- pages 1004–1005. genitor endothelial cells for angiogenesis. Science 275: 964–967, 1997 27. Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, This article contains supplemental material online at http://jasn.asnjournals. Croker BP, Demetris AJ, Drachenberg CB, Fogo AB, Furness P, Gaber org/lookup/suppl/doi:10.1681/ASN.2013121277/-/DCSupplemental.

J Am Soc Nephrol 26: 1161–1171, 2015 Endothelial Cell Reactive Antibody 1171 Supplementary Table 1. Patient Demographics

ELISA Test Discovery Cohort Cohort N=10 N=150 Recipient Age (mean, SD) 58 ± 9 49 ± 15 Race (% non-white) 10% 44%

Male Gender (%) 90% 41% Previous transplantation (%) 60% 40% HLA Sensitization1 (%) 90% 91% 2 Mean % CPRA (CDC-XM, FCXM) 18, 38 29, 39 3 Original ABO or HLA barrier : (%) ABOi 20% 6% CDC-XM+ 0 2% FCXM+ 10% 19% FCXM-, DSA+ 40% 45% NO DSA 50% 34% Donor (Mean Age) 45 ± 12 40 ± 15 Live Donor (%) 70% 55% Deceased Donor (%) 30% 45% HLA-A;B;DR;DQ mismatch (mean) 5.0 4.7 Plasmapheresis Treatments: No Pre- or Post- Treatments (%) 20% 45% Pre-transplant (Mean, Median) 2.5, 1.5 1.0, 0.0 Post-transplant (Mean, Median) 4.4, 3.5 4.0, 2.0

anti-CD25 induction (% ) 40% 17%

Thymoglobulin induction (% ) 60% 83%

Rituximab induction (%) 30% 36%

1 HLA-specific antibody detected on Luminex® platforms 2 2 Calculated panel reactive antibody (CPRA) was determined for HLA-antibodies of sufficient strength to yield a positive CDC crossmatch (CDC-XM) or flow cytometric crossmatch (FCXM) 3 Original donor HLA-specific antibody (DSA) strength prior to desensitization treatments

Supplementary Table 2. Protein array analysis of 14 AECA eluates derived from 10 Discovery Cohort Recipients Protein Name Gene Average Symbol RFU 1. Recombinant human CTLA-4/Fc CTLA-4 58077.0 2. tripartite motif-containing 21 (TRIM21) TRIM21 39835.7 3. hematopoietic SH2 domain containing (HSH2D) HSH2D 28572.5 4. interferon, alpha-inducible protein 6 (IFI6) IFI6 15892.2 5. APEX nuclease (apurinic/apyrimidinic endonuclease) 2 (APEX2), APEX2 12493.3 nuclear gene encoding mitochondrial protein 6. CAP-GLY domain containing linker protein family, member 4 (CLIP4) CLIP4 10955.7 7. UBX domain containing 8 (UBXD8) UBXD8 8636.5 8. zinc finger, MYM-type 5 (ZMYM5) ZMYM5 8260.8 9. EGF-like repeats and discoidin I-like domains 3 (EDIL3) EDIL3 8232.8 10. ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) UBE2V1 7955.9 11. phosphoglycerate dehydrogenase (PHGDH) PHGDH 7405.2 12. sciellin (SCEL) SCEL 7255.3 13. Zinc finger CCHC domain-containing protein 8 ZCCHC8 5908.2 14. chromosome 22 open reading frame 33 (C22orf33) C22orf33 5578.2 15. cleavage and polyadenylation specific factor 3, 73kDa (CPSF3) CPSF3 5425.6 16. Uncharacterized protein C20orf96 C20orf96 5031.8 17. endoglin (Osler-Rendu-Weber syndrome 1) (ENG) ENG 4966.8 18. Disks large homolog 3 DLG3 4352.0 19. cyclin G associated kinase (GAK) GAK 4298.4 20. CDC42 effector protein (Rho GTPase binding) 3 (CDC42EP3) CDC42EP3 4280.3 21. ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1), ARFGAP1 4211.4 transcript variant 2 22. baculoviral IAP repeat-containing 4 (BIRC4) BIRC4 4108.5 23. chromosome 2 open reading frame 47 (C2orf47) C2orf47 3597.3 24. quaking homolog, KH domain RNA binding (mouse) (QKI), transcript QKI 3493.8 variant 4 25. intercellular adhesion molecule 4 (Landsteiner-Wiener blood ICAM4 3412.4 group) (ICAM4), transcript variant 1 26. fms-related tyrosine kinase 3 ligand (FLT3LG) FLT3LG 3383.8 27. hypothetical protein LOC51233, mRNA (cDNA clone MGC:75009 LOC51233 3331.7 IMAGE:5170001), complete cds. 28. Parkinson disease 7 domain containing 1 (PDDC1) PDDC1 3296.6 29. forkhead box P1 (FOXP1) FOXP1 3267.5 30. nudix (nucleoside diphosphate linked moiety X)-type motif 16-like 1 NUDT16L1 3256.8 (NUDT16L1) 31. ubiquilin 2 (UBQLN2) UBQLN2 3129.8 32. TOX high mobility group box family member 2 (TOX2), transcript TOX2 3102.1 variant 3 33. AF4/FMR2 family, member 4 (AFF4) AFF4 3050.3

34. CWF19-like 2, cell cycle control (S. pombe) (CWF19L2) CWF19L2 2924.5 35. glutaryl-Coenzyme A dehydrogenase (GCDH), nuclear gene GCDH 2666.8 encoding mitochondrial protein, transcript variant 1 36. Protein FAM184A C6orf60 2661.1 37. SMT3 suppressor of mif two 3 homolog 1 (S. cerevisiae) (SUMO1), SUMO1 2559.8 transcript variant 1 38. chromogranin B (secretogranin 1) (CHGB) CHGB 2559.3 39. zinc finger protein 695 (ZNF695) ZNF695 2529.8 40. LIM homeobox transcription factor 1, alpha (LMX1A) LMX1A 2476.6 41. complexin 2 (CPLX2), transcript variant 2 CPLX2 2435.8 42. myotilin (MYOT) MYOT 2362.6 43. sorting nexin 13 (SNX13) SNX13 2324.0 44. intercellular adhesion molecule 4 (Landsteiner-Wiener blood ICAM4 2268.7 group) (ICAM4) 45. centrosome and spindle pole associated protein 1 (CSPP1), CSPP1 2256.9 transcript variant 2 46. glycogenin 2 (GYG2) GYG2 2227.8 47. outer dense fiber of sperm tails 2 (ODF2) ODF2 2226.6 48. transcriptional adaptor 3 (NGG1 homolog, yeast)-like (TADA3L), TADA3L 2204.3 transcript variant 2 49. aquaporin 2 (collecting duct) (AQP2) AQP2 2197.4 50. dystrophin (muscular dystrophy, Duchenne and Becker types) DMD 2136.8 (DMD), transcript variant Dp71b 51. hypothetical protein FLJ22795 (FLJ22795) FLJ22795 2117.2 52. muted homolog (mouse) (MUTED) MUTED 2096.1 53. proline/arginine-rich end leucine-rich repeat protein (PRELP), PRELP 2063.3 transcript variant 1 54. major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1) HLA-DPA1 2053.5 55. testis-specific serine kinase 2 (TSSK2) TSSK2 2046.3