Serum Donor-specific Antibodies Research Articles

Memory B-cell derived donor-specific antibodies do not predict outcome in sensitized kidney transplant recipients: a retrospective single-center study.

Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan-Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.

Tracking Circulating HLA-Specific IgG-Producing Memory B Cells with the B-Cell ImmunoSpot Assay.

Donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules are a major risk factor for rejection of transplanted organs (in antibody-mediated rejection [ABMR]), particularly in patients who have prior sensitization or receive insufficient immunosuppression through minimization or noncompliance. These DSA are measured routinely in the serum of patients prior to transplantation mainly using bead-based technologies or cell-based assays. However, the absence of detectable serum DSA does not always reflect the absence of sensitization or histologically defined ABMR, and so it has been proposed that the detection and measurement of memory B cells capable of secreting antibodies against donor HLA antigens could be carried out using B-cell ImmunoSpot, to better inform the degree of immune sensitization of transplant patients prior to as well as after transplantation. Such an assay is described here.

Morphologic and immunophenotypic evaluation of liver allograft biopsies with contemporaneous serum DSA measurements.

Acute antibody mediated rejection is increasingly identified in liver allografts as a unique form of alloimmune injury associated with donor specific antibodies (DSA). This manifests pathologically as microvascular injury and C4d uptake. Despite the liver allograft's relative resistance to alloimmune injury, liver allografts are not impervious to cellular and antibody-mediated rejection. In this blinded control study, we evaluated CD163 immunohistochemistry and applied the Banff 2016 criteria for diagnosis of acute AMR on a group of indication allograft liver biopsies from DSA positive patients and compared them to indication biopsies from DSA negative controls. Most DSA positive patients were females (75%, p=.027), and underwent transplantation for HCV infection. Significant histopathological predictors of serum DSA positivity were Banff H-score (p=.01), moderate to severe cholestasis (p=.03), and CD163 score>2 (p=.029). Other morphologic features that showed a trend with DSA positivity include Banff portal C4d-score (p=.06), bile ductular reaction (p=.07), and central perivenulitis (p=.07). The odds of DSA sMFI ≥5000 was 12.5 times higher in those with a C4d score>1 than those with aC4d score≤1 (p=.04). Incidence of definite for aAMR in the DSA positive cohort was 25% (n=5), and 0% in the DSA negative cohort. A group of 5 DSA positive cases were not classifiable by the current scheme. Sinusoidal CD163, Banff H-score, and diffuse C4d are predictors of serum DSA, and facilitate recognition of histopathological features associated with serum DSA and tissue-antibody interaction.

Congenic hematopoietic stem cell transplantation promotes survival of heart allografts in murine models of acute and chronic rejection.

The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin-Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Antibodies against complement component C5 prevent antibody-mediated rejection after lung transplantation in murine orthotopic models with skin-graft-induced pre-sensitization

ObjectiveAntibody-mediated rejection (AMR) could induce acute or chronic graft failure during organ transplantation. Several reports have shown that anti-C5 antibodies are effective against AMR after kidney transplantation. However, few reports have assessed the efficacy of anti-C5 antibodies against AMR after lung transplantation. Therefore, this study aimed to evaluate the efficacy of this novel therapy against AMR after lung transplantation.MethodsBALB/c and C57BL/6 mice were used as donors and recipients. One group was pre-sensitized (PS) by skin transplantation 14 days before lung transplantation. The other group was non-sensitized (NS). Orthotopic left-lung transplantation was performed in both groups. Animals were killed at 2 or 7 days after lung transplantation and evaluated for histopathology, C4d immunostaining, and serum donor-specific antibodies (DSAs) (n = 5 per group). Isograft (IS) models with C57BL/6 mice were used as controls. To evaluate the efficacy of C5 inhibition, other animals, which received similar treatments to those in the PS group, were treated with anti-C5 antibodies, cyclosporine/methylprednisolone, anti-C5 antibodies/cyclosporine/methylprednisolone, or isotype-matched irrelevant control monoclonal antibodies (n = 5 per group).ResultsTwo days after lung transplantation, the NS group exhibited mild, localized graft-rejection features (rejection score: 0.45 ± 0.08, p = 0.107). The PS group exhibited AMR features with a significantly higher rejection score (2.29 ± 0.42, p = 0.001), C4d vascular-endothelium deposition, and substantial presence of serum DSA. On day 7 after lung transplantation, both groups showed extensive graft alveolar wall destruction, and high acute-rejection scores. Mice receiving anti-C5 antibodies or anti-C5/antibodies/cyclosporine/methylprednisolone demonstrated significantly lower acute-rejection scores (0.63 ± 0.23, p = 0.002; 0.59 ± 0.22, p = 0.001, respectively) than those receiving isotype control antibodies.ConclusionsMurine orthotopic allograft lung transplant models met the clinical diagnosis and pathogenesis classification criteria of AMR. In these models, anti-C5 antibodies suppressed AMR. Therefore, anti-C5 therapy may be effective against AMR after lung transplantation.

Donor-Derived Cell-Free DNA Plus Tissue Gene Expression Profiling of Lung Allograft Injury

<h3>Purpose</h3> Lung transplantation outcomes are limited by chronic lung allograft dysfunction, of which acute rejection is a significant risk factor. Acute rejection includes acute cellular rejection (ACR) and antibody-mediated rejection (AMR), and historically has been diagnosed through a combination of transbronchial biopsy histopathology, pulmonary function testing, and the presence of serum donor-specific antibodies. Donor-derived cell-free DNA (dd-cfDNA) has recently shown promise as a less invasive biomarker of allograft injury, but, in isolation, dd-cfDNA does not sufficiently delineate ACR from AMR from infection. Here we propose to combine dd-cfDNA analysis of allograft injury with in situ lung tissue gene expression to better characterize different forms of allograft injury following lung transplantation. <h3>Methods</h3> This is a single center retrospective cohort study of at least 20 lung transplant recipients with longitudinally matched plasma and transbronchial biopsies (n=100). All samples have previously been collected and stored. Plasma will be obtained from the institution's Total Transplant Care Protocol (TTCP) Biorepository, while formalin fixed paraffin embedded (FFPE) biopsy specimens will be obtained from the Department of Pathology through the institution's Tissue Archive Service (TAS). Samples will be assigned to specific clinical cohorts based on histopathology, radiology, microbiological studies and pulmonary function tests at the time of sample collection. Plasma dd-cfDNA fractions will be quantified using Allosure Lung (CareDx), and tissue gene expression profiles (GEP) will be captured with the nCounter Gene Expression Assay (Nanostring Technologies). Data will be analyzed with non-parametric tests and multivariate analyses to determine associations between dd-cfDNA levels and tissue gene expression patterns. <h3>Endpoints</h3> 1. Correlate plasma dd-cfDNA levels with acute allograft injury from ACR, AMR, or infection and determine the time course of dd-cfDNA expression during the first year after lung transplantation. 2. Validate the nCounter Gene Expression Assay platform and identify gene(s) associated with different types of acute rejection and/or infection.

Lactic Acidosis, Hypoglycemia, and Eosinophilia: Novel Markers of Antibody-Mediated Rejection Causing Graft Ischemia.

Historically, minimal significance has been given to the humoral immune response after liver transplantation and, despite antibodies against HLA antigens being present in 10-20% of patients awaiting transplant, the incidence of antibody mediated rejection (AMR) after ABO compatible (ABO-C) liver transplantation is low (1).Donor Specific Antibodies (DSA) can lead to early graft loss (2), but most patients awaiting liver transplant are not screened for HLA sensitisation and serum DSA measurement are only undertaken if AMR is suspected. We describe two cases of AMR occurring within one week of ABO-C liver transplantation with previously unreported biochemical and histological characteristics.

Cyclic Helix B Peptide Prolongs Skin Allograft Survival via Inhibition of B Cell Immune Responses in a Murine Model.

Antibody-mediated rejection (AMR) represents a major cause of allograft dysfunction and results in allograft failure in solid organ transplantation. Cyclic helix B peptide (CHBP) is a novel erythropoietin-derived peptide that ameliorated renal allograft rejection in a renal transplantation model. However, its effect on AMR remains unknown. This study aimed to investigate the effect of CHBP on AMR using a secondary allogeneic skin transplantation model, which was created by transplanting skin from BALB/c mice to C57BL/6 mice with or without CHBP treatment. A secondary syngeneic skin transplantation model, involving transplantation from C57BL/6 mice to C57BL/6 mice, was also created to act as a control. Skin graft rejection, CD19+ B cell infiltration in the skin allograft, the percentages of splenic plasma cells, germinal center (GC) B cells, and Tfh cells, the serum levels of donor specific antibodies (DSAs), and NF-κB signaling in splenocytes were analyzed. Skin allograft survival was significantly prolonged in the CHBP group compared to the allogeneic group. CHBP treatment also significantly reduced the CD19+ B cell infiltration in the skin allograft, decreased the percentages of splenic plasma cells, GC B cells, and Tfh cells, and ameliorated the increase in the serum DSA level. At a molecular level, CHBP downregulated P100, RelB, and P52 in splenocytes. CHBP prolonged skin allograft survival by inhibiting AMR, which may be mediated by inhibition of NF-κB signaling to suppress B cell immune responses, thereby decreasing the DSA level.

Complement Inhibition as Potential Novel Therapy for Antibody-Mediated Rejection after Lung Transplantation in Murine Antibody-Mediated Rejection Model

Antibody-mediated rejection (AMR) can induce acute or chronic graft failure in organ transplantations. There are few reports regarding AMR in lung transplant, and no reports of effective therapy for AMR after lung transplant. This study aimed to verify the efficacy of anti-complement 5(anti-C5), which is reported to prevent AMR in kidney transplant, in an AMR-enhanced mouse model after lung transplant. As allograft model, BALB/c (Major histocompatibility complex; MHC H2b) and C57BL/6 (MHC H2b) were used for donor and recipient in order to detect and enhance AMR. Skin transplants were performed to pre-sensitize, and subsequently, orthotopic left lung transplants were performed 14 days after skin transplant. The mice were treated with anti-C5 (40mg/kg/day) (n=5), or cyclosporine (CyA, 10mg/kg/day) /methylprednisolone (mPSL, 1.6mg/kg/day) (n=5), or anti-C5/CyA/mPSL (n=5), or with isotype-matched irrelevant control mAb (n=5) subcutaneously until sacrifice. The mice were sacrificed 2 days after lung transplant and evaluated for histopathology, including acute rejection score, C4d immunostaining, and donor-specific antibody (DSA) in serum. The mice with anti-C5 showed significantly lower C5 in serum compared with the mice with isotype control (1472.50±110.44 vs. 2081.87±75.17 pg/ml, p=0.002). The mice treated with anti-C5 and anti-C5/CyA/mPSL showed significantly lower rejection A score (0.63±0.23; p=0.002, 0.59±0.22; p=0.001) compared with the mice with isotype control (2.58±0.35). All groups showed C4d deposition on vascular endothelium and DSA in serum. Anti-C5 effectively prevented AMR against transplanted allogeneic lung in the pre-sensitized murine model, in which AMR was strongly augmented.

RNA Expression Analysis of Antibody Mediated Rejection after Heart Transplant with or without Donor Specific Antibodies

Antibody mediated rejection (AMR) is an increasingly studied cause of graft failure after heart transplantation. AMR diagnostic criteria previously required serum donor specific antibodies (DSA). However, the most recent criteria only requires pathological findings independent of DSA. This classification defined a subset of patients with AMR without known antibodies. To investigate this complication, we performed RNA sequencing using endomyocardial biopsy samples taken at the time of pathology confirmed AMR in the presence and absence of DSA. We evaluated the transcriptome profile of the paraffin-embedded endomyocardial biopsy specimens of 30 representative heart transplant recipients. Patients were categorized as having no evidence of AMR (controls), immunohistochemical findings of AMR (pAMR1i) in the presence of DSA (1i+D), pAMR1i in the absence of DSA (1i-D), histopathological findings of AMR (pAMR1h) in the presence of DSA (1h+D), pAMR1h in the absence of DSA (1h-D) or presence of both immunohistochemical and histopathological findings of AMR (pAMR2) in the presence of DSA. A total of 5 patients were analyzed per group. All were matched for previous mechanical circulatory support and time from transplant to biopsy. There was no differential RNA expression between patients with no AMR and those in the 1i-D group. There was also no differential expression between patients in the 1h+D and pAMR2 groups. The largest difference was seen between the no AMR and 1i+D groups and the no AMR and pAMR2 groups. In addition, there was RNA expression differences between patients with immunohistochemical evidence of AMR compared to those with histopathological findings, suggesting distinct mechanisms. These studies demonstrate that RNA expression profiling can be performed on endomyocardial biopsy tissue preserved in paraffin from the time of a rejection episode and that different expression patterns are observed dependent on the criteria used to diagnose AMR. In particular, AMR in the presence of DSA showed a different RNA expression profile than AMR without DSAs. The RNA expression associated with histological markers of AMR also appeared different than that associated with immunological markers. Overall, our findings suggest the existence of different subtypes of AMR that may rely on distinct mechanisms of injury.

Intragraft donor-specific anti-HLA antibodies in phenotypes of chronic lung allograft dysfunction.

Circulating anti-human leukocyte antigen (HLA) serum donor-specific antibodies (sDSAs) increase the risk of chronic lung allograft dysfunction (CLAD) and mortality. Discrepancies between serological and pathological/clinical findings are common. Therefore, we aimed to assess the presence of tissue-bound graft DSAs (gDSAs) in CLAD explant tissue compared with sDSAs. Tissue cores, obtained from explant lungs of unused donors (n=10) and patients with bronchiolitis obliterans syndrome (BOS; n=18) and restrictive allograft syndrome (RAS; n=18), were scanned with micro-computed tomography before elution of antibodies. Total IgG levels were measured via ELISA. Anti-HLA class I and II IgG gDSAs were identified using Luminex single antigen beads and compared with DSAs found in serum samples. Overall, mean fluorescence intensity was higher in RAS eluates compared with BOS and controls (p<0.0001). In BOS, two patients were sDSA+/gDSA+ and two patients were sDSA-/gDSA+. In RAS, four patients were sDSA+/gDSA+, one patient was sDSA+/gDSA- and five patients were sDSA-/gDSA+. Serum and graft results combined, DSAs were more prevalent in RAS compared with BOS (56% versus 22%; p=0.04). There was spatial variability in gDSA detection in one BOS patient and three RAS patients, who were all sDSA-. Total graft IgG levels were higher in RAS than BOS (p<0.0001) and in gDSA+ versus gDSA- (p=0.0008), but not in sDSA+ versus sDSA- (p=0.33). In RAS, total IgG levels correlated with fibrosis (r= -0.39; p=0.02). This study underlines the potential of gDSA assessment as complementary information to sDSA findings. The relevance and applications of gDSAs need further investigation.

Development of Novel Murine Antibody Mediated Rejection Model after Orthotopic Lung Transplant

Purpose Antibody mediated rejection (AMR) has been playing an important role in organ transplantation inducing acute or chronic graft failure, which directly influence the survival of graft. There is little knowledge about AMR in lung transplant compared with other organ. Animal disease model is warranted to elucidate mechanism and development of novel drug for AMR, hence our purpose of this study is to establish murine AMR model of lung transplant. Methods As allograft model, BALB/c (Major histocompatibility complex; MHC H2b) and C57BL/6 (MHC H2b) were used for donor and recipient in order to detect and enhance AMR. Study groups were divided into two groups, including pre-sensitized by skin transplant (PS) group (n=10) and non-sensitized (NS) group (n=10). Skin transplant was performed 14days before lung transplant only in PS group, and orthotopic left lung transplant were performed in both groups. They were sacrificed at 2day or 7day post lung transplant, and evaluated for histopathology including acute rejection score, C4d immunostaining, and donor specific antibody (DSA) in serum. Isograft model (IS) using C57/BL6 murine was used for control. Results NS group showed only localized rejection features on their graft and rejection score (0.36±0.13, p=0.13; Fig a, b) at day 2 after lung transplant, on the other hand, PS group showed clinical features of AMR, including high rejection score (2.19±0.27, p=0.003), C4d deposition on vascular endothelium, and DSA in serum at day 2 after lung transplant. On day 7 after lung transplant, the both groups showed extensive destruction of alveolar wall in graft, high acute rejection score (NS 3.25±0.18, p Conclusion The murine orthotopic allograft lung transplant model satisfied criteria of AMR. The allograft model pre-sensitized by skin transplant showed enhanced AMR at early period post lung transplant; therefore this model could be useful to examine pathogenesis of AMR and development of new therapy for AMR.

Intragraft IgG Levels and Donor-Specific anti-HLA Antibodies in Different Phenotypes of Chronic Lung Allograft Dysfunction

Purpose Circulating donor specific antibodies (DSA) are a key diagnostic criterion for antibody-mediated rejection (AMR) after lung transplantation (LTx). DSA occur in 10-50% of LTx patients and increase the risk of chronic lung allograft dysfunction (CLAD) and mortality. Detection and interpretation of circulating DSA remains unstandardized and challenging; and discrepancies between serological and pathological/clinical findings are common. Therefore, we aimed to assess presence and relevance of graft DSA (gDSA) in explant lungs with different phenotypes of CLAD. Methods Explant lungs from patients with bronchiolitis obliterans syndrome (BOS, n= 18) and restrictive allograft syndrome (RAS, n=18) were collected at redo-transplantation or autopsy, inflated and fixed in liquid nitrogen fumes. Per lung, two tissue cores were selected from separate regions, assessed for surface density using microCT, and subsequently homogenized. Intragraft IgG antibodies were eluted (Gamma ELU-kit II, Immucor) and total IgG levels were measured via ELISA (Thermo Fisher). Anti-HLA Class I and II antibodies were identified via Luminex (LABScreen LS1A04 and LS2A01, One Lambda). gDSA data was correlated to serum DSA (sDSA) profiles, assessed for all patients included in this study using routine clinical practice. Results Intragraft IgG levels were higher in RAS compared to BOS samples (p Conclusion IgG levels were higher in RAS and in gDSA+ samples. DSA are more prevalent in RAS compared to BOS. gDSA assessment could provide complementary information to sDSA findings. The relevance and applications of gDSA need further investigation.

Value of monitoring circulating donor-reactive memory B cells to characterize antibody-mediated rejection after kidney transplantation.

Antibody-mediated rejection (ABMR) is defined by specific histopathological lesions and evidence of circulating donor-specific antibodies (DSA). Although DSA are not always detectable, monitoring donor-reactive memory B cells (mBC) could identify patients at risk of developing ABMR. Peripheral donor-reactive mBC using a novel HLA B cell ELISpot assay, serum DSA, and numbers of different B cell subsets were assessed in 175 consecutive kidney transplants undergoing either for-cause or 6- and 24-month surveillance biopsies for their association with main histological lesions of ABMR and impact on allograft outcome. In 85 incident for-cause biopsies, high frequencies of donor-reactive mBC were detected in all 16 (100%) acute ABMR/DSA+ and most chronic ABMR, with or without DSA (24/30[80%] and 21/29[72.4%], respectively). In a longitudinal cohort of 90 nonsensitized patients, a progressively higher expansion of donor-reactive mBC than de novo DSA was observed at 6 and 24 months (8.8% vs 7.7% and 15.5% vs 11.1%, respectively) and accurately identified patients with ongoing subclinical ABMR (area under the curve=0.917 and area under the curve=0.809, respectively). An unsupervised hierarchical cluster analysis revealed a strong association between donor-reactive mBC with main fundamental allograft lesions associated with ABMR and conferred a significant deleterious impact on graft outcome. Monitoring donor-reactive mBC may be useful to further characterize humoral rejection after kidney transplantation.

Kidney transplant in sensitized patients: A case series from a premier teaching hospital in Malaysia

BackgroundKidney transplantation is associated with improved survival as opposed to long-term dialysis in end-stage renal disease (ESRD) patients. In Malaysia, due to the limited number of cadaveric and compatible living related renal transplant (LRRT), there is a need to explore the potential of transplant with ABO-incompatible and positive donor specific antibodies (DSA). This report describes a case series of sensitized patients undergoing kidney transplant. ObjectiveThe aim of this study is to assess the immediate graft function (IGF), graft functions at the 1st month, 3rd month, 6th month and 12th month post-transplant. Graft functions were assessed using serum creatinine, protocol biopsy (at 1st, 3rd, 6th and 12th months post-transplant) and DSA level. MethodsData was collected from all sensitized kidney transplant done at the University Malaya Medical Center (UMMC) between February 2016 and April 2017. ABO-incompatible kidney transplant will be excluded from this study. The patient's progress and outcome (kidney function) are fully documented. ResultsFive patients were analyzed for this report. The first two patients developed antibody-mediated rejection (ABMR) immediately, post-transplant. Learning from initial experience, we have adopted our own desensitization protocol and subsequent to that, all three patients had IGF. Three out of five patients have borderline rejection on protocol biopsy but did not require pulse with methylprednisolone or intensification of immunosuppression. Three patients had negative preformed DSAs at 3-month post-transplant. ConclusionDesensitization protocols may help to overcome incompatibility barriers in live-donor renal transplantation. Based on our experiences we have adopted a protocol that comprises of intravenous rituximab, plasma exchange and intravenous thymoglobulin.

369 Therapeutic Plasma Exchange in Small-Bowel Transplant Recipients With Acute Allograft Rejection

Therapeutic plasma exchange (TPE) is used in solid-organ transplant patients to treat antibody-mediated rejection (AMR) by removing donor specific antibodies (DSAs). While ASFA guidelines exist for treating AMR in other solid-organ transplants, no such guidelines exist for small bowel-allograft patients; the benefit of TPE in AMR of small bowel transplant patients has not been studied. Five patients were followed with serial surveillance ileoscopies and allograft biopsies. Tissues were processed using standard protocols (UNMC) for permanent histologic examination. Two H&E stained slides from each biopsy were examined microscopically by gastrointestinal/transplant pathologists for evidence of ACR. To assess for AMR, serial serum DSA levels were obtained via by Luminex LabScreen single antigen bead assay (One Lambda, Canoga Park, CA). Patients were treated with at least five TPE procedures. To determine whether TPE provided benefit, we examined three parameters in the post-apheresis period: DSA presence and strength, histopathologic rejection, and clinical response. All patients developed class I and/or class II DSAs following transplant. No clear change in DSA levels following TPE was seen. Sixty percent of patients (3/5) had histopathologic evidence of acute cellular rejection (ACR) prior to initiation of TPE. Following TPE, biopsies showed no evidence of ACR or did not meet the criteria necessary for evaluation. Management of rejection in small bowel transplant patients, caused by both antibody-mediated and T-cell driven processes, is extremely complex. We examined the impact of performing TPE in patients with histologic and/or clinical evidence of acute rejection. A clear trend in the burden or activity of DSAs, histopathologic improvement of allograft biopsies, or clinical improvement following administration of TPE, was not seen. Thus, in our study, TPE does not appear to have a clear benefit in the treatment of AMR and/or ACR in small bowel rejection. Additional studies will be needed to corroborate our results.

P002 Patient outcomes following liver-kidney transplantation with donor specific antibodies

Aim Simultaneous liver and kidney transplantation (SLKT) remains as the gold standard for patients with chronic renal and liver diseases. Pre-transplant donor-specific antibody (DSA) to class I antigens have been shown to decrease following SLKT; whereas, DSA to class II antigens may remain detrimental to long term graft survival. Methods We analyzed a cohort of SLKT performed at our institution from 2011 to 2017. We ran both flow and cytotoxic crossmatches pre-transplant and crossmatch positive samples were rerun in the post transplant period. We monitored for DSA both pre and post transplant using a single antigen bead assay. (One Lambda). Results There were 30 SLKT performed, of which 11 had pre-formed DSA (four class I and II DSA, five class I DSA only, two class II only). Strong donor specific anti-HLA antibodies were eliminated following SLKT for class I and II specificities while preserving anti-HLA antibodies against antigens that were not expressed on the donor. The presence of DSA lead to six positive flow cytometry crossmatches of which three were cytotoxic crossmatch positive. Repeat crossmatching with recipient serum post SLKT was found to be negative which correlated with the sharp decrease in DSA MFI detectable in recipient serum. Whether DSA in recipient serum was against class I, II or both antigens there was no significant difference in one-year patient or graft survival. Conclusions The liver absorbed the impact of DSA in our cohort of SLKT recipients. The SLKT recipient did well following transplant in the presence of DSA to class I, II or both. Given the high priority SLK patients receive, having strong DSA no matter the class with a positive XM result should not be a contraindication to transplant.

Inhibition of Spleen Tyrosine Kinase Reduces Renal Allograft Injury in a Rat Model of Acute Antibody-Mediated Rejection in Sensitized Recipients.

Organ transplantation into sensitized patients with preexisting donor-specific antibodies (DSA) is very challenging. Spleen tyrosine kinase (Syk) promotes leukocyte recruitment and activation via signaling through various cell surface receptors. We investigated whether a selective Syk inhibitor (GS-492429) could suppress antibody-mediated rejection (AMR) in a rat model of AMR in sensitized recipients. Recipient Lewis rats (RT1) were immunized with donor (Dark Agouti, RT1) spleen cells (day -5). Recipients underwent bilateral nephrectomy and orthotopic renal transplantation (day 0). Cellular rejection was minimized by tacrolimus treatment from day -1. Groups received GS-492429 (30 mg/kg, twice a day) (n = 11) or vehicle (n = 12) from 1 hour before transplantation until being killed on day 3. Vehicle-treated recipients developed graft dysfunction on day 1 which rapidly worsened by day 3. Histology showed severe damage (thrombosis, acute tubular injury, capillaritis) and infiltration of many Syk leukocytes. GS-492429 did not affect graft dysfunction on day 1, but treatment reduced allograft damage and prevented the rapid deterioration of graft function on day 3. GS-492429 reduced the prominent macrophage infiltrate and reduced the M1 proinflammatory response. Neutrophil and NK cell infiltration and capillary thrombosis were also significantly reduced by GS-492429 treatment. Serum DSA levels and the deposition of IgG and C4d in the allograft were equivalent in the 2 groups. Treatment with a Syk inhibitor significantly reduced renal allograft injury in a model of severe antibody-mediated damage in highly sensitized recipients. Further studies are warranted to determine whether Syk inhibition is a potential adjunctive treatment in clinical AMR.

Refinement of the criteria for ultrastructural peritubular capillary basement membrane multilayering in the diagnosis of chronic active/acute antibody-mediated rejection.

Chronic active/acute antibody-mediated rejection (cABMR) is the main cause of late renal allograft loss. Severe peritubular capillary basement membrane multilayering (PTCML) assessed on electron microscopy is one diagnostic feature of cABMR according to the Banff 2013 classification. We aimed to refine the PTCML criteria for an earlier diagnosis of cABMR. We retrospectively investigated ultrastructural features of 159 consecutive renal allografts and 44 nonallografts. The presence of serum donor-specific antibodies at the time of biopsy of allografts was also examined. Forty-three patients (27.0%) fulfilled the criteria of cABMR, regardless of PTCML, and comprised the cABMR group. Forty-one patients (25.8%) did not exhibit cABMR features and comprised the non-cABMR allograft control group. In addition, 15 zero-day wedge resections and 29 native kidney biopsies comprised the nonallograft control group. When the diagnostic accuracies of various PTCML features were assessed using the cABMR and non-cABMR allograft control groups, ≥4 PTCML, either circumferential or partial, in ≥2 peritubular capillaries of the three most affected capillaries exhibited the highest AUC value (0.885), greater than the Banff 2013 classification (0.640). None of the nonallograft control groups exhibited PTCML features. We suggest that ≥4 PTCML in ≥2 peritubular capillaries of the three most affected cortical capillaries represents the proper cutoff for cABMR.

P074 Donor specific anti-HLA antibodies are associated with subclinical allograft injury in heart transplant recipients

Aim Anti-HLA antibodies detected after heart transplantation have been associated with graft rejection and worse survival. However, the presence of donor specific antibodies (DSA) alone is not diagnostic of antibody mediated rejection (AMR). To better define the significance of circulating DSA, we investigated the relationship between DSA and relevant histo- and immunopathologic features of endomyocardial biopsies. Methods Sixty-three patients received heart transplantation between Jan 1st 2011 and Dec 31st 2013 and were followed at our center. Serum anti-HLA antibodies were monitored using the Single Antigen Bead assay (One Lambda, Canoga Park, CA). Endomyocardial biopsies were performed serially during the first year post-transplant and yearly thereafter, according to the existing clinical protocol. Results At a median followup time of 33 months, graft/patient survival was 98%. Sixteen (25%) out of 63 patients had at least 2 DSA positive tests after transplantation. DSA were predominantly directed to HLA class II antigens, with 13 out of 16 patients displaying anti-HLA-DQ DSA. A total of 906 endomyocardial biopsies were performed after transplantation. Out of 906, 305 biopsies had concomitant DSA results and were included in the analysis. Fifty-seven (19%) biopsies had positive DSA associated results. ACR was diagnosed in 60 (20%) out of 305 biopsies. There was no significant correlation between ACR and serum DSA. AMR was diagnosed in one (0.3%) biopsy, which had a concomitant positive DSA. The following histo- and immunopathologic parameters were analyzed in relation to DSA: perivascular inflammation, interstitial inflammation, myocyte injury, lymphocytic infiltration, interstitial edema, interstitial hemorrhage, fibrosis, microvascular changes and C4d staining. Microvascular changes included prominent endothelial cells and intravascular mononuclear cells and/or neutrophils. DSA were associated with microvascular changes (N = 9; p Conclusion Our results provide evidence that circulating DSA are associated with subclinical graft injury. Post-transplant DSA monitoring may serve as a useful, non-invasive tool to identify patients at risk for early graft loss.