Presence Of Donor-specific Antibodies Research Articles

Follow-up biopsies with microvascular inflammation and persistent donor specific antibodies identify ongoing rejection in pediatric kidney transplant recipients.

Inadequate treatment of acute rejection (AR) in pediatric kidney transplant recipients (KTR) can contribute to early allograft failure. Serum creatinine is an insensitive marker of allograft function, especially in the pediatric population, and may not detect ongoing rejection after treatment. We evaluated the utility of follow-up biopsies to detect persistent inflammation and future episodes of rejection. We performed a single-center retrospective review to identify pediatric KTR with biopsy-proven rejection and a subsequent follow-up biopsy, noting type of AR, Banff scores, serum creatinine, and presence of donor specific antibodies (DSA). Outcomes included resolution of AR, change in eGFR, DSA, persistent microvascular inflammation (MVI) and future episodes of AR. Twelve cases of cellular (TCMR), 9 antibody-mediated (AMR), and 8 mixed cases of AR were identified among 23 KTR. Resolution was noted in 75% with TCMR, significantly higher than AMR (22%) or mixed rejection (13%), p < 0.01. Those without resolution of AR on follow-up biopsy were more likely to have ongoing episodes of AR or graft loss (p = 0.02). Persistence of DSA and MVI was associated with lack of AR resolution (p = 0.01 and p = 0.001, respectively). Those with persistent MVI on follow-up biopsy had higher probability of future AR events or graft loss, p = 0.003. Follow-up biopsies to assess response to AR treatment revealed that most cases of TCMR were successfully treated but that AMR and mixed rejection portend a componentof chronicity and more complicated course. Identification of persistent subclinical inflammation predicts future rejection episodes, has adverse effects on graft longevity, and can inform the need for additional treatment. We advocate for implementation of a follow-up biopsy protocol and future study of non-invasive biomarkers paired with protocol biopsies.

Abstract 4140154: Highly Sensitized Heart Transplant Recipients Who Have Undergone Pre-Transplant Desensitization Therapies Demonstrate Acceptable Medium-Term Outcomes

Introduction/Research Question: Allosensitization, the presence of circulating anti-HLA antibodies, is a barrier in heart transplantation (HT), restricting the donor pool size, and leading to increased waitlist mortality and rejection risk post-HT. Desensitization therapies can be used to broaden the donor pool in highly sensitized patients, defined as pre-HT calculated panel reactive antibodies (cPRA)≥50%, for antibodies with mean fluorescence intensity (MFI)&gt;10,000. There is paucity of data on longer-term outcomes in such high-immunological risk patients. Methods: Sensitized patients with pre-HT cPRA&gt;50%, who were treated with desensitization and then received HT between 2011-2022 at Cedars-Sinai Medical Center were included. Desensitization therapies included bortezomib, rituximab, tocilizumab, obinutuzumab, intravenous immunoglobulin (IVIG), and plasmapheresis. cPRA, donor-specific antibody (DSA) levels, and post-HT clinical outcomes were assessed up to 5-year follow-up, loss to follow-up, or death. Results: 40 patients were analyzed. 77.5% of patients were female, and all had at least 1 risk factor for sensitization. cPRA decreased from 84.5±13.5% at baseline to 74.1±22.4% after completion of desensitization therapy, prior to transplant (p=0.005). Mean follow-up time post-HT was 4.3±2.9 years. 45.0% of patients had antibody-mediated rejection (AMR), 12.5% had CAV. Overall survival was 94.9%, 92.1%, and 87.5% at 1, 3, and 5 years respectively. All patients except 1 had normal left ventricular function at last follow-up. 72.5% of patients were transplanted in the presence of DSA, and 60.0% underwent post-HT induction with eculizumab in addition to antithymocyte globulin (ATG)/IVIG, while the remaining received ATG/IVIG alone. Among HT recipients with pre-formed DSA, 41.4% had high-level DSA (MFI &gt; 10,000) at time of HT. At 6-12 months post-HT, only 17.2% had high-level DSA, and 41.4% had resolution of DSA. Conclusion: Highly sensitized HT candidates who underwent pre-HT desensitization had comparable survival and CAV rates as compared to the general HT population reported in the literature. However, sensitized patients experienced higher rates of AMR which were not associated with graft dysfunction or mortality.

Progress in Noninvasive Surveillance for Acute Rejection in Pediatric Heart Transplant Recipients: A Real-World Analysis of Donor-Derived Cell-Free DNA-Based Surveillance Protocol.

Acute cellular (ACR) and antibody-mediated (AMR) rejection are risk factors for allograft loss in heart transplant (HT) recipients. Endomyocardial biopsy (EMB), although considered the gold standard for rejection surveillance, is invasive and has high interobserver variability. Noninvasive donor-derived cell-free DNA (dd-cfDNA) sampling has a high negative predictive value (NPV) for rejection in adults and is increasingly used in pediatrics. This single center study aimed to test the performance of dd-cfDNA in screening for acute rejection (AR) and donor-specific antibodies (DSAs) in pediatric HT recipients. Blood samples for dd-cfDNA were obtained per clinical protocol for all eligible HT recipients in our center from July 1, 2022 to December 31, 2023. Primary endpoints were episodes of AR, pathology grading of EMBs temporally related to ddcfDNA sampling (0-150 days), and presence of DSAs. There were 471 interpretable samples, in 192 unique patients. Of those, 199 dd-cfDNA tests were paired with EMB ± DSA in 152 patients. Abnormal dd-cfDNA (> 0.2%) was found in 77 samples (median 0.48%, range 0.21%-11%) and led to EMB, where one sample was positive for ACR (grade 2R), 13 for AMR, yielding an NPV of 97% for AMR. After excluding abnormal ddcfDNA testing associated with AR, 65 abnormal dd-cfDNA tests were paired with DSA testing. The NPV of the test for detection of DSAs was 93%. Implementation of noninvasive rejection surveillance with dd-cfDNA in a pediatric cohort demonstrates high NPV for AR and high DSAs, making it an ideal screening tool for long-term monitoring of allograft health in pediatrics.

Antibody-mediated rejection diagnosed in early protocol biopsies in high immunological risk kidney transplant recipients.

Renal transplant recipients with donor-specific anti-HLA antibodies are at an increased risk of antibody-mediated rejection (AMR). Early protocolized renal biopsies may serve as a strategy to improve diagnosis in this patient population. We evaluated 155 highly sensitized renal transplant recipients with cPRA class I+II>90% pre-transplant from 2015 to 2022. Patients with protocol biopsies within the first two weeks post-transplant were included. A total of 122 patients were included in the study. Of these, 13 (10.6%) were diagnosed with very early antibody-mediated rejection (veABMR) within the first two weeks post-transplant. This corresponds to 52% (13/25 patients) of all ABMR cases reported during the follow-up of this population. The graft survival rates at one and three years were significantly lower in patients with veABMR (p<0.001) compared to patients without rejection in the early protocol biopsy. In terms of severity, the veABMR cohort exhibited a hazard ratio (HR) of 10.33 (95% CI 3.23-33.06; p<0.001) for graft failure. The presence of donor-specific antibodies (DSA) class II on the day of transplantation and a higher percentage of eplet mismatch (EpMM), particularly EpMM DQA1, correlated with the development of veABMR. Early protocol biopsies play a pivotal role in the early detection of veABMR in high-risk immunological patients. Patients with veABMR face significant risks of graft loss, despite early treatment of rejection.

Chimeric HLA antibody receptor T cell therapy for humoral transplant rejection.

Antibody-mediated rejection (ABMR) is a significant obstacle to achieving optimal long-term outcomes after solid organ transplantation. The presence of donor-specific antibodies (DSAs), particularly against human leucocyte antigen (HLA), increases the risk of allograft rejection and subsequent graft loss. No effective treatment for ABMR currently exists, warranting novel approaches to target the HLA-specific humoral alloimmune response. Cellular therapies may hold promise to this end. According to publicly available sources as of now, three independent laboratories have genetically engineered a chimeric HLA antibody receptor (CHAR) and transduced it into human T cells, based on the demonstrated efficacy of chimeric antigen receptor T cell therapies in malignancies. These CHAR-T cells are designed to exclusively eliminate B cells that produce donor-specific HLA antibodies, which form the cornerstone of ABMR. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA-specific B cells, sparing B cells with other specificities. Thus CHAR technology may be used as a selective desensitization protocol and to treat ABMR after solid organ transplantation.

Functionalized magnetic nanoparticles remove donor-specific antibodies (DSA) from patient blood in a first ex vivo proof of principle study

The presence of donor-specific antibodies (DSA) such as antibodies directed against donor class I human leucocyte antigen (e.g., HLA-A) is a major barrier to kidney transplant success. As a proof of concept, functionalized magnetic nanoparticles have been designed to eliminate DSA from saline, blood and plasma of healthy donors and sensitized patients. Specific HLA-A1 protein was covalently bound to functionalized cobalt nanoparticles (fNP), human serum albumin (HSA) as control. fNP were added to anti-HLA class I-spiked saline, spiked volunteers’ whole blood, and to whole blood and plasma of sensitized patients ex vivo. Anti-HLA-A1 antibody levels were determined with Luminex technology. Antibodies' median fluorescent intensity (MFI) was defined as the primary outcome. Furthermore, the impact of fNP treatment on blood coagulation and cellular uptake was determined. Treatment with fNP reduced MFI by 97 ± 2% and by 94 ± 4% (p < 0.001 and p = 0.001) in spiked saline and whole blood, respectively. In six known sensitized anti-HLA-A1 positive patients, a reduction of 65 ± 26% (p = 0.002) in plasma and 65 ± 33% (p = 0.012) in whole blood was achieved. No impact on coagulation was observed. A minimal number of nanoparticles was detected in peripheral mononuclear blood cells. The study demonstrates—in a first step—the feasibility of anti-HLA antibody removal using fNP. These pilot data might pave the way for a new personalized DSA removal technology in the future.

#3036 The influence of first renal graft on second graft rejection and survival: the importance of revisiting histocompatibility and clinical outcomes

Abstract Background and Aims Kidney retransplantation confers a robust survival benefit over dialysis and recent data has shown similar outcomes to first kidney transplant. The number of HLA mismatches is an important predictor of graft loss and sensitization and the presence of donor specific antibodies (DSA) -pre and -post transplant is a major risk factor for rejection and worst outcomes. However, the importance of HLA matching of first graft and the relevance of first donor DSAs on second graft outcomes is less known. Our aim was to identify risk factors to second graft acute rejection and graft loss, related to second and first graft features. Method We performed a retrospective, longitudinal study including all patients submitted to a 2nd kidney transplant between January 2008 and December 2021, excluding patients with more than 2 grafts or multi-organ transplant. Clinical and histocompatibility data from the first and second donor and recipient were collected. HLA antibodies were detected by solid phase assays and considered if MFI &amp;gt;2500. For subsequent kidney transplants, repeated mismatches are not allowed and unacceptable antigens are those for which the patient has pre-formed -A, -B and -DR antibodies, with MFI&amp;gt;1000. Biopsy proven acute rejection was defined according to Banff 2017 criteria. Follow-up was 31st December 2023 for functioning grafts or time to graft failure, with a mean time of 92 ± 53 months. Results We included 114 patients, 73 (64%) males, mostly Caucasians (96%), with a mean age of 43.9 ± 13 years at 2nd transplant. First kidney transplant was performed before 2010 for 99 patients (87%), with a median first graft survival of 82.5 [12.5-144] months and 19 patients (16.7%) presented primary disfunction due to surgical/vascular complications. After graft loss, 43 patients (37.7%) had their graft removed. For second transplant, 11 (9.6%) patients had a living donor. Induction therapy with thymoglobulin occurred in 94 patients (83%) and maintenance therapy with mycophenolic acid, calcineurin inhibitors and steroids was prescribed in 83 patients (73%). During follow-up, 20 patients (17.5%) presented biopsy proven acute rejection, 13 (65%) T cell mediated and 7 (35%) antibody-mediated, the majority during the first-year post-transplant (N = 17, 85%). The risk factors for second graft rejection are summarized in Table 1. Patients with a first graft nephrectomy had a higher risk of acute rejection (OR 3.9, 95% CI [1.4-10.9], p &amp;lt; 0.006) and were more susceptible to the development of second donor post-transplant DSA (58% vs 35%, p = 0.02). Death censored second allograft loss occurred in 24 patients (21%) at follow up and acute rejection was an important risk factors (OR 4.3, 95% CI [1.5-12.1], p = 0.004). First-year graft survival was 93% and a survival at follow up was 79%. On survival analysis the presence of second graft DSA was a major risk factor for graft loss, especially class 2 -DQ and -DR. First transplant total mismatch load, especially on class 1 contributed to worst graft survival. Conclusion Worst outcomes in first kidney transplant were related with an increased risk of acute rejection in second graft and 1st graft nephrectomy increases the risk of the development of 2nd graft DSA and acute rejection. First transplant mismatch load decreased second graft survival.

The Predictors and Risk Factors of 2-Year Rejection in Renal Transplant Patients: A Multicenter Case-Control Study

Introduction: Kidney transplantation is a definitive treatment for end-stage renal disease. It is associated with improved life expectancy and quality of life. One of the most common complications following kidney transplantation is graft rejection. To our knowledge, no previous study has identified rejection risk factors in kidney transplant recipients in Saudi Arabia. Therefore, this study aimed to determine the specific risk factors of graft rejection. Methods: A multicenter case-control study was conducted at four transplant centers in Saudi Arabia. All adult patients who underwent a renal transplant between January 1, 2015 and December 31, 2021 were screened for eligibility. Included patients were categorized into two groups (cases and control) based on the occurrence of biopsy-proven rejection within 2 years. The primary outcome was to determine the risk factors for rejection within the 2 years of transplant. Exact matching was utilized using a 1:4 ratio based on patients’ age, gender, and transplant year. Results: Out of 1,320 screened renal transplant recipients, 816 patients were included. The overall prevalence of 2-year rejection was 13.9%. In bivariate analysis, deceased donor status, the presence of donor-specific antibody (DSA), intraoperative hypotension, Pseudomonas aeruginosa, Candida, and any infection within 2 years were linked with an increased risk of 2-year rejection. However, in the logistic regression analysis, the presence of DSA was identified as a significant risk for 2-year rejection (adjusted OR: 2.68; 95% CI: 1.10, 6.49, p = 0.03). Furthermore, blood infection, infected with Pseudomonas aeruginosa or BK virus within 2 years of transplant, were associated with higher odds of 2-year rejection (adjusted OR: 3.10; 95% CI: 1.48, 6.48, p = 0.003, adjusted OR: 3.23; 95% CI: 0.87, 11.97, p = 0.08 and adjusted OR: 2.76; 95% CI: 0.89, 8.48, p = 0.07, respectively). Conclusion: Our findings emphasize the need for appropriate prevention and management of infections following kidney transplantation to avoid more serious problems, such as rejection, which could significantly raise the likelihood of allograft failure and probably death. Further studies with larger sample sizes are needed to investigate the impact of serum chloride levels prior to transplant and intraoperative hypotension on the risk of graft rejection and failure.

Molecular diagnosis of antibody-mediated rejection: Evaluating biopsy-based transcript diagnostics in the presence of donor-specific antibodies but without microvascular inflammation, a single-center descriptive analysis

Biopsy-based transcript diagnostics may identify molecular antibody-mediated rejection (AMR) when microvascular inflammation (MVI) is absent. In this single-center cohort, biopsy-based transcript diagnostics were validated in 326 kidney allograft biopsies. A total of 71 histological AMR and 35 T cell–mediated rejection (TCMR) cases were identified as molecular AMR and TCMR in 55% and 63%, respectively. Among 121 cases without MVI (glomerulitis + peritubular capillaritis = 0), 45 (37%) donor-specific antibody (DSA)-positive and 76 (63%) DSA-negative cases were analyzed. Twenty-one out of the 121 (17%) cases showed borderline changes, or TCMR, while BK nephropathy was excluded. None of the 45 DSA-positive patients showed molecular AMR. Among 76 DSA-negative patients, 2 had mixed molecular AMR/TCMR. All-AMR phenotype scores (sum of R4-R6) exhibited median values of 0.13 and 0.12 for DSA-positive and DSA-negative patients, respectively (P = .84). A total of 13% (6/45) DSA-positive and 11% (8/76) DSA-negative patients showed an all-AMR phenotype score > 0.30 (P = .77). Patients with a higher all-AMR phenotype score showed 33% more histologic TCMR (P = .005). The median all-AMR phenotype scores of glomerular basement membrane double contours = 0 and glomerular basement membrane double contours > 0 biopsies were 0.12 and 0.10, respectively (P = .35). Biopsy-based transcript diagnostics did not identify molecular AMR in cases without MVI. Follow-up biopsies and outcome data should evaluate the clinical relevance of subthreshold molecular alterations.

A modern heart transplant rejection surveillance protocol utilizing cell-free DNA: A single-center experience

INTRODUCTIONEndomyocardial biopsy (EMBx) is considered the gold standard for rejection monitoring after heart transplantation; however, it is invasive and histologic interpretation has limitations. Sensitive blood biomarkers including donor-derived cell-free DNA (dd-cfDNA) have emerged to decrease EMBx frequency. METHODSWe retrospectively reviewed data on 237 patients who underwent heart transplantation at our institution. Of these, 125 patients underwent monitoring using dd-cfDNA, combined with a fewer number of EMBx, and 112 patients underwent monitoring using EMBx only. We compared rates of rejection, graft dysfunction, and survival at 1 year. RESULTSMedian age at time of transplant was 59.8 years, and 77.6% were men. In the dd-cfDNA group, there were significantly fewer episodes of EMBx defined ACR (2.5% vs. 18.8%, P<0.001) and treated ACR (4.2% vs. 19.6%, P=0.001). Comparatively, there were more EMBx defined AMR (5% vs. 0.9%) and treated AMR (5% vs. 2.7%) in the dd-cfDNA group. No significant differences were observed in graft dysfunction, presence of donor-specific antibodies, or survival at 1 year. CONCLUSIONSIn conclusion, a modern rejection surveillance protocol utilizing non-invasive testing is safe, led to significantly fewer EMBx, fewer treated rejection episodes, and no difference in survival at 1 year. More AMR episodes identified via dd-cfDNA could lead the way for more accurate diagnostic and treatment decisions.

Waiting time for kidney transplantation based on calculated panel reactive antibodies: experience of a southern Brazilian center.

The aim of this study was to analyze the waiting list for kidney transplantation in our hospital according to candidate's panel reactive antibodies (cPRA) and its outcomes. One thousand six hundred forty patients who were on the waiting list between 2015 and 2019 were included. For the analysis, hazard ratios (HR) for transplant were estimated by Fine and Gray's regression model according to panel reactivity and HR for graft loss and death after transplantation. The mean age was 45.39 ± 18.22 years. Male gender was predominant (61.2%), but the proportion decreased linearly with the increase in cPRA (p < 0.001). The distribution of patients according to panels were: 0% (n = 390), 1% - 49% (n = 517), 50% - 84% (n = 269), and ≥ 85% (n = 226). Transplantation was achieved in 85.5% of the sample within a median time of 8 months (CI 95%: 6.9 - 9.1). The estimated HRs for transplantation during the follow-up were 2.84 (95% CI: 2.51 - 3.34), 2.41(95%CI: 2.07 - 2.80), and 2.45(95%CI: 2.08 - 2.90) in the cPRA range of 0%, 1%-49%, and 50%-84%, respectively, compared to cPRA ≥ 85 (p < 0.001). After transplantation, the HR for graft loss was similar in the different cPRA groups, but the HR for death (0.46 95% CI 0.24-0.89 p = 0.022) was lower in the 0% cPRA group when adjusted for age, gender, and presence of donor specific antibodies (DSA). Patients with cPRA below 85% are more than twice as likely to receive a kidney transplantation with a shorter waiting time. The risk of graft loss after transplantation was similar in the different cPRA groups, and the adjusted risk of death was lower in nonsensitized recipients.

Superiority of Single-Antigen Bead Study in Donor-Specific Antibodies: Determination in Highly Sensitized Patients.

The presence of donor-specific antibodies against HLA before kidney transplant has been variably associated with decreased long-term graft survival. Data on the association between pretransplant donor-specific antibodies and rejection and cause of graft failure in recipients of donor kidneys are scarce. For this study of HLA antibody levels, we analyzed serum samples from 76 patients (48 women and 28 men) who were prepared for kidney transplant at the Baskent University İstanbul Hospital between 2017 and 2022. Levels were determined by using Lifecodes panel reactive antibody class I and II identification kits and Lifecodes LSA class I and II identification kits by the Luminex assay method. Multiple antigen tests showed more than 70% sensitization detected against both class I and class II antigens in our patient group. When some samples were reevaluated with the single-antigen bead method, desensitization values were shown to be considerably reduced compared with values from multiple antigen methods. The single-antigen-coated bead method can be useful in determining the risk of donor-specific antibodies in highly sensitized patients.

Allogeneic Bone Marrow Transplantation from HLA-Identical or Haploidentical Donors for Children and Adolescents with Sickle Cell Disease: A Feasible Curative Option

The only curative option widely available for sickle cell disease (SCD) is bone marrow transplantation (BMT). Since few patients have a healthy matched sibling donor (MSD), haploidentical (haplo) donors have expanded the donor pool to approximately 90% of the patients. The addition of thiotepa to the Johns Hopkins conditioning therapy backbone dor haplo BMT showed durable engraftment and excellent overall survival. However, graft rejection has remained an important obstacle. Our challenge in these patients has been to augment the conditioning therapy to provide adequate engraftment, while maintaining a low transplant-related mortality. Methods: Retrospective data of all 26 patients with SCD followed by our group after MSD (n=7) and haplo (n=19) BMT performed in four different institutions. With a MSD, conditioning was rATG 4.5mg/kg, Busulfan (Bu, AUC 4,500), Fludarabine (Flu, 120mg/m²) or rATG, Bu, Cyclophosphamide (Cy, 200mg/kg) and with haplo donors, rATG 4.5mg/kg, Thiotepa 10mg/kg, Flu 150mg/m², Cy 29 mg/kg and TBI 2Gy. Patients were prepared to BMT with intense hypertransfusion/bleeding or erythrocytapheresis to keep HbS&amp;lt;30 and Hb~10g/dL, reticulocytes &amp;lt;10% for 2-3 months prior to BMT, with intensive chelation to keep ferritin &amp;lt;1,000ng/mL. The presence of donor specific antibodies would exclude the patient from undergoing BMT. Due to two consecutive secondary graft failures (no detectable donor cells despite initial engraftment) and two falling mixed chimerism among the first 13 patients undergoing haplo, the doses of the conditioning therapy were increased to Cy 50 mg/kg and TBI 4Gy in a single fraction, as previously used in our patients with aplastic anemia undergoing BMT. Six consecutive patients received this augmented conditioning therapy. All patients had serial chimerism measured by STR. The graft-versus-host disease (GVHD) prophylaxis included Cyclosporine - Methotrexate in MSD and post-transplant Cy/mesna, mycophenolate mofetil and sirolimus in haplo. Results: Between September 2016 and April 2023, 26 patients underwent BMT, with a median age of 11 years (4-20), half of them females, 7 with a MSD and 19 an haplo (4 siblings, 6 fathers, and 9 mothers); 77% of the donors had sickle cell trait. The graft was bone marrow in all but one patient with Sb thal and Hodgkin's lymphoma. The median follow-up was 15.5 months. All patients engrafted. BMT with MSD had excellent outcomes and stable chimerism. Among the first 13 patients undergoing haplo, secondary graft failure occurred very suddenly in two patients (CD34 cell doses of 2 and 8x10 6 CD34/kg), both male teenagers; two additional patients had a significant drop in chimerism up to 30% donor cells but responded to serial donor leukocyte infusions 1-5 x10 6 CD3/kg (2 and 6) and developed very mild skin GVHD. The other patients maintained &amp;gt; 89-100% donor cells. Thereafter, with the augmented conditioning regimen, all six consecutive patients have stable 100% donor chimerism. Acute II-III GVHD was observed in 34% and chronic GVHD in 15% of the patients. Viral reactivations (CMV, HHV6, BKV, HHV7 and EBV) were observed in all patients. Overall survival is 100% and rejection-free survival is 83% at 1 year (Figures 1, 2). Conclusions: Allogeneic BMT with an HLA-identical or haploidentical family donor was feasible to treat SCD with an excellent overall survival. With a larger cohort and longer follow-up, the long-term effects of this strategy will be thoroughly studied.

The Molecular Microscope Diagnostics System (MMDx) Does Not Identify Early Molecular Antibody-Mediated Rejection (ABMR) in the Presence of Donor-Specific Antibodies (DSA), but Absence of Microvascular Inflammation

The Molecular Microscope Diagnostics System (MMDx) Does Not Identify Early Molecular Antibody-Mediated Rejection (ABMR) in the Presence of Donor-Specific Antibodies (DSA), but Absence of Microvascular Inflammation

Plasma NGAL levels in stable kidney transplant recipients and the risk of allograft loss.

The objective of this study was to investigate the utility of neutrophil gelatinase-associated lipocalin (NGAL) and calprotectin (CPT) to predict long-term graft survival in stable kidney transplant recipients (KTR). A total of 709 stable outpatient KTR were enrolled >2 months post-transplant. The utility of plasma and urinary NGAL (pNGAL, uNGAL) and plasma and urinary CPT at enrollment to predict death-censored graft loss was evaluated during a 58-month follow-up. Among biomarkers, pNGAL showed the best predictive ability for graft loss and was the only biomarker with an area under the curve (AUC)>0.7 for graft loss within 5 years. Patients with graft loss within 5 years (n=49) had a median pNGAL of 304 [interquartile range (IQR) 235-358] versus 182 (IQR 128-246) ng/mL with surviving grafts (P<.001). Time-dependent receiver operating characteristic analyses at 58 months indicated an AUC for pNGAL of 0.795, serum creatinine-based Chronic Kidney Disease Epidemiology Collaboration estimated glomerular filtration rate (eGFR) had an AUC of 0.866. pNGAL added to a model based on conventional risk factors for graft loss with death as competing risk (age, transplant age, presence of donor-specific antibodies, presence of proteinuria, history of delayed graft function) had a strong independent association with graft loss {subdistribution hazard ratio (sHR) for binary log-transformed pNGAL [log2(pNGAL)] 3.4, 95% confidence interval (CI) 2.24-5.15, P<.0001}. This association was substantially attenuated when eGFR was added to the model [sHR for log2(pNGAL) 1.63, 95% CI 0.92-2.88, P=.095]. Category-free net reclassification improvement of a risk model including log2(pNGAL) in addition to conventional risk factors and eGFR was 54.3% (95% CI 9.2%-99.3%) but C-statistic did not improve significantly. pNGAL was an independent predictor of renal allograft loss in stable KTR from one transplant center but did not show consistent added value when compared with baseline predictors including the conventional marker eGFR. Future studies in larger cohorts are warranted.

Targeting TFH cells is a novel approach for donor-specific antibody desensitization of allograft candidates: an in vitro and in vivo study.

The presence of donor-specific antibodies (DSA) are associated with graft failure either following human leukocyte antigen (HLA)-mismatched allogeneic stem cell transplantation or after organ transplantation. Although targeting B cells and plasma cells have been used for desensitization, there have been reports of failure. T-follicular helper (Tfh) cells assist B cells in differentiating into antibody-secreting plasma cells. We used haploidentical allograft as a platform to investigate the possibility of targeting Tfh cells to desensitize DSA. The quantities of circulating Tfh (cTfh) cell subsets in allograft candidates were abnormal, and these cells, including the cTfh2 and cTfhem cell subsets, were positively related to the production of anti-HLA antibodies. Ex vivo experiments showed that the cTfh cells of anti-HLA antibody-positive allograft candidates could induce B cells to differentiate into DSA-producing plasmablasts. The immune synapse could be involved in the assistance of cTfh cells to B cells in antibody production. In vitro experiments and in vivo clinical pilot studies indicated that targeting cTfh cells with sirolimus can inhibit their auxiliary function in assisting B cells. Ex vivo and in vivo studies demonstrated the effect of sirolimus and rituximab on DSA desensitization compared with either sirolimus or rituximab alone (60%, 43.75%, and 30%, respectively). Our findings provide new insight into the role of Tfh cells in the pathogenesis of DSA production in HLA-mismatched transplant candidates. Our data also indicate that targeting Tfh cells is a novel strategy for DSA desensitization and combination of sirolimus and rituximab might be a potential therapy. The prospective cohort of this study is registered at http://www.chictr.org.cn as #ChiCTR-OPC-15006672.

Preventing kidney transplant failure by screening for antibodies against human leucocyte antigens followed by optimised immunosuppression: OuTSMART RCT

Design Investigator-led, prospective, open-labelled marker-based strategy (hybrid) randomised trial. Background Allografts in 3% of kidney transplant patients fail annually. Development of antibodies against human leucocyte antigens is a validated predictive biomarker of allograft failure. Under immunosuppression is recognised to contribute, but whether increasing immunosuppression can prevent allograft failure in human leucocyte antigen Ab+ patients is unclear. Participants Renal transplant recipients &gt; 1 year post-transplantation attending 13 United Kingdom transplant clinics, without specific exclusion criteria. Interventions Regular screening for human leucocyte antigen antibodies followed, in positive patients by interview and tailored optimisation of immunosuppression to tacrolimus, mycophenolate mofetil and prednisolone. Objective To determine if optimisation of immunosuppression in human leucocyte antigen Ab+ patients can cost-effectively prevent kidney allograft failure. Outcome Time to graft failure after 43 months follow-up in patients receiving the intervention, compared to controls, managed by standard of care. Costs and quality-adjusted life-years were used in the cost-effectiveness analysis. Randomisation and blinding Random allocation (1 : 1) to unblinded biomarker-led care or double-blinded standard of care stratified by human leucocyte antigen antibodies status (positive/negative) and in positives, presence of donor-specific antibodies (human leucocyte antigen antibodies against donor human leucocyte antigen) or not (human leucocyte antigen antibodies against non-donor human leucocyte antigen), baseline immunosuppression and transplant centre. Biomaker-led care human leucocyte antigen Ab+ patients received intervention. Human leucocyte antigen Ab-negative patients were screened every 8 months. Recruitment Began September 2013 and for 37 months. The primary endpoint, scheduled for June 2020, was moved to March 2020 because of COVID-19. Numbers randomised From 5519 screened, 2037 were randomised (1028 biomaker-led care, 1009 to standard of care) including 198 with human leucocyte antigen antibodies against donor human leucocyte antigen (106 biomaker-led care, 92 standard of care) and 818 with human leucocyte antigens antibodies against non-donor human leucocyte antigen (427 biomaker-led care, 391 standard of care). Numbers analysed Two patients were randomised in error so 2035 were included in the intention-to-treat analysis. Outcome The trial had 80% power to detect a hazard ratio of 0.49 in biomarker-led care DSA+ group, &gt; 90% power to detect hazard ratio of 0.35 in biomarker-led care non-DSA+ group (with 5% type 1 error). Actual hazard ratios for graft failure in these biomarker-led care groups were 1.54 (95% CI: 0.72 to 3.30) and 0.97 (0.54 to 1.74), respectively. There was 90% power to demonstrate non-inferiority of overall biomarker-led care group with assumed hazard ratio of 1.4: This was not demonstrated as the upper confidence limit for graft failure exceeded 1.4: (1.02, 95% CI 0.72 to 1.44). The hazard ratio for biopsy-proven rejection in the overall biomarker-led care group was 0.5 [95% CI: 0.27 to 0.94: p = 0.03]. The screening approach was not cost-effective in terms of cost per quality-adjusted life-year. Harms No significant differences in other secondary endpoints or adverse events. Limitations Tailored interventions meant optimisation was not possible in some patients. We did not study pathology on protocol transplant biopsies in DSA+ patients. Conclusions No evidence that optimised immunosuppression in human leucocyte antigen Ab+ patients delays renal transplant failure. Informing patients of their human leucocyte antigen antibodies status appears to reduce graft rejection. Future work We need a better understanding of the pathophysiology of transplant failure to allow rational development of effective therapies. Trial registration This trial is registered as EudraCT (2012-004308-36) and ISRCTN (46157828). Funding This project was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation programme (11/100/34) and will be published in full in Efficacy and Mechanism Evaluation; Vol. 10, No. 5. See the NIHR Journals Library website for further project information.

Chimeric HLA antibody receptor T cells for targeted therapy of antibody-mediated rejection in transplantation.

The presence of donor-specific antibodies (DSA), mainly against HLA, increases the risk of allograft rejection. Moreover, antibody-mediated rejection (ABMR) remains an important barrier to optimal long-term outcomes after solid organ transplantation. The development of chimeric autoantibody receptor T lymphocytes has been postulated for targeted therapy of autoimmune diseases. We aimed to develop a targeted therapy for DSA desensitization and ABMR, generating T cells with a chimeric HLA antibody receptor (CHAR) that specifically eliminates DSA-producing B cells. We have genetically engineered an HLA-A2-specific CHAR (A2-CHAR) and transduced it into human T cells. Then, we have performed in vitro experiments such as cytokine measurement, effector cell activation, and cytotoxicity against anti-HLA-A2 antibody-expressing target cells. In addition, we have performed A2-CHAR-Tc cytotoxic assays in an immunodeficient mouse model. A2-CHAR expressing T cells could selectively eliminate HLA-A2 antibody-producing B cells in vitro. The cytotoxic capacity of A2-CHAR expressing T cells mainly depended on Granzyme B release. In the NSG mouse model, A2-CHAR-T cells could identify and eradicate HLA-A2 antibody-producing B cells even when those cells are localized in the bone marrow. This ability is effector:target ratio dependent. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA class I antibody-producing B cells. Thus, we consider that CHAR technology may be used as a selective desensitization protocol or an ABMR therapy in transplantation.

#5691 BK POLYOMAVIRUS VIREMIA AND VIRURIA ARE ASSOCIATED WITH SEVERE INFLAMMATION AND FIBROSIS IN RENAL ALLOGRAFT, BUT NOT WITH GRAFT SURVIVAL

Abstract Background and Aims Infection of the renal allograft by Polyomavirus BK (BKV) causes tubular cell injury and interstitial inflammation, which leads to subsequent interstitial fibrosis and tubular atrophy (IFTA). The presence and degree of inflammation within these areas (i-IFTA) is associated with worse outcomes in kidney transplant (KT) patients. The aim of this study was to investigate the association between BKV viremia and viruria and the presence of IFTA and i-IFTA, decline of renal function, and graft loss. Method We conducted a single-center retrospective case-control study, which included all patients who underwent an allograft biopsy between January 2018 and December 2022. All biopsies were made for-cause (allograft dysfunction with increased serum creatinine). Real-time polymerase chain reaction was used to detect and quantitate BK viral load in serum and urine samples. Patients were included if they had at least 6 months since KT and if they had serial evaluation of BKV viremia and viruria previously to the biopsy. We evaluated, in the graft biopsy, the percentage of IFTA, and also i-IFTA. Estimated glomerular filtration rate (eGFR) was obtained at baseline and 1 year after the biopsy. Graft loss was evaluated at 6 months and 1 year after the biopsy. Results Mean age of the population was 53,5 years and 67% were males. A total of 116 biopsies were performed during the studied period and 72 met the inclusion criteria. Ten percent of patients developed persistent (≥ 3 months) BKV viremia, 24% developed persistent BKV viruria, and 18% developed persistent JCV viruria. Five cases of polyomavirus-associated nephropathy were diagnosed. The other most common diagnoses were T cell-mediated rejection (24%), antibody-mediated rejection (15%), and focal segmental glomerulosclerosis (15%). In patients with persistent BKV viremia, a higher viral load had a strong positive association with the degree of i-IFTA (r=0.954; P = .001). Patients with BKV viremia and/or viruria were more likely to present more severe IFTA (≥ 40%) and i-IFTA (grade ≥2) in allograft biopsy (P = .03). Patients with higher degrees of IFTA and i-IFTA were more likely to experience graft loss 1 year after the biopsy (P = .01). There was no significant association between the presence of BKV viremia and/or viruria and the decline of eGFR, nor with the incidence of graft loss 1 year after the biopsy. In multivariable analysis adjusted for age, eGFR at the time of biopsy, the presence of donor-specific antibodies and the main diagnosis of allograft biopsy, the presence of BK viremia and/or viruria was a significant predictor of more severe IFTA and i-IFTA (P = .04). Conclusion In our population, higher BK viremia loads were associated with more extensive inflammation within areas of IFTA. Patients with BK viremia and/or viruria, independently of the main diagnosis of the biopsy, were more likely to exhibit more severe IFTA and i-IFTA in allograft biopsies, but no association with the incidence of dialysis at 1 year was shown. Longer prospective studies are needed to investigate whether BKV infection treatment/ reduction of immunosuppression can slow down fibrosis progression and minimizes i-IFTA.

Asymptomatic Allograft Fibrosis in Pediatric Liver Transplantation: Potential Clinical Implications.

Asymptomatic Allograft Fibrosis in Pediatric Liver Transplantation: Potential Clinical Implications.