Clinical Antibody-mediated Rejection Research Articles

Pseudomonas aeruginosa infection correlates with high MFI donor-specific antibody development following lung transplantation with consequential graft loss and shortened CLAD-free survival

BackgroundDonor-specific antibodies (DSAs) are common following lung transplantation (LuTx), yet their role in graft damage is inconclusive. Mean fluorescent intensity (MFI) is the main read-out of DSA diagnostics; however its value is often disregarded when analyzing unwanted post-transplant outcomes such as graft loss or chronic lung allograft dysfunction (CLAD). Here we aim to evaluate an MFI stratification method in these outcomes.MethodsA cohort of 87 LuTx recipients has been analyzed, in which a cutoff of 8000 MFI has been determined for high MFI based on clinically relevant data. Accordingly, recipients were divided into DSA-negative, DSA-low and DSA-high subgroups. Both graft survival and CLAD-free survival were evaluated. Among factors that may contribute to DSA development we analyzed Pseudomonas aeruginosa (P. aeruginosa) infection in bronchoalveolar lavage (BAL) specimens.ResultsHigh MFI DSAs contributed to clinical antibody-mediated rejection (AMR) and were associated with significantly worse graft (HR: 5.77, p < 0.0001) and CLAD-free survival (HR: 6.47, p = 0.019) compared to low or negative MFI DSA levels. Analysis of BAL specimens revealed a strong correlation between DSA status, P. aeruginosa infection and BAL neutrophilia. DSA-high status and clinical AMR were both independent prognosticators for decreased graft and CLAD-free survival in our multivariate Cox-regression models, whereas BAL neutrophilia was associated with worse graft survival.ConclusionsP. aeruginosa infection rates are elevated in recipients with a strong DSA response. Our results indicate that the simultaneous interpretation of MFI values and BAL neutrophilia is a feasible approach for risk evaluation and may help clinicians when to initiate DSA desensitization therapy, as early intervention could improve prognosis.

Imlifidase in Highly Sensitized Kidney Transplant Recipients With a Positive Crossmatch Against a Deceased Donor

IntroductionImlifidase is authorized for desensitization of highly sensitized adult kidney transplant candidates with a positive crossmatch against a deceased donor. Here, we report on the results for the first 9 patients transplanted in this context who had at least 3 months of follow-up. MethodsThe eligibility criteria were: calculated panel reactive antibodies (cPRA) ≥ 98%, ≥ 3 years on the waiting list, immunodominant donor specific antibodies (iDSA) with mean fluorescence intensity (MFI) > 6,000 (and < 5,000 at 1:10 dilution) and a negative post-imlifidase complement-dependent cytotoxic crossmatch (CDCXM). ResultsAll 9 patients had been on dialysis for an average of 123±41 months, with cPRA at 99% (n=2) or 100% (n=7). At transplantation, the mean number of DSA was 4.3±1.4. The median iDSA MFI was 9,153 (6,430-16,980). Flow cytometry and CDC XMs before imlifidase were positive in 9 and 2 patients, respectively. After one injection of imlifidase all were negative. Patients received polyclonal antibodies, intravenous immunoglobulins, rituximab, tacrolimus, and mycophenolate. Five patients had a DSA rebound within the first 14 days: 2 had concomitant clinical antibody-mediated rejection (ABMR), 2 had subclinical ABMR, and 1 had isolated positive C4d staining. No ABMR was observed in patients without rebound. CKD-EPI eGFR was 56±22 mL/min/1.73m2 at the last follow-up (7±2.8 months). No graft loss or death were observed. Four patients developed at least one infection. ConclusionThese real-life data demonstrate that the use of imlifidase to desensitize highly sensitized patients can have an acceptable short-term efficacy and safety profile in selected patients.

Oxidized ATP Suppresses B Lymphocyte Activity to Attenuate Antibody-mediated Rejection of Kidney Allografts in Mice

Background. Antibody-mediated rejection (AMR) is a major cause of renal allograft dysfunction and loss. Targeting B cells and/or donor-specific antibody removal using plasma exchange and anti-CD20 antibodies are increasingly used in clinical practice, but the efficacy remains limited. Recent studies suggest that targeting purinergic P2X7 receptor/ATP axis can have profound immune regulatory effects in transplant models, but the mechanisms involved remain incompletely defined. Methods. Purified B cells were isolated from the spleen of Balb/C mice and cultured with oxidized ATP at different concentrations. Proliferation and differentiation of B cells were examined. Effects of oxidized ATP were examined in a presensitized animal model where kidney allograft rejection mimics aspects of clinical AMR. Histopathology was assessed at the time of rejection or on day 5 after kidney transplantation. Infiltrating immune cells in renal allografts were detected by flow cytometry. Results. Oxidized ATP inhibited B-cell activation and proliferation in vitro, significantly attenuated histological signs of graft injury and prolonged kidney allograft survival. Mechanistically, oxidized ATP inhibited antibody secretion by activated B cells in response to lipopolysaccharide stimulation and markedly suppressed the production of donor-specific antibody in kidney allograft recipients. Oxidized ATP also reduced graft infiltration by other inflammatory cells. Conclusions. These findings provide evidence for the involvement of the purinergic P2X7 receptor pathway in AMR and suggest that targeting this pathways may have important clinical implications.

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.

Clinical features and allograft failure rates of pulmonary antibody-mediated rejection categories.

Pulmonary antibody-mediated rejection (AMR) consensus criteria categorize AMR by diagnostic certainty. This study aims to define the clinical features and associated outcomes of these recently defined AMR categories. Adjudication committees reviewed clinical data of 335 lung transplant recipients to define clinical or subclinical AMR based on the presence of allograft dysfunction, and the primary endpoints, time from transplant to allograft failure, a composite endpoint of chronic lung allograft dysfunction and/or death. Clinical AMR was subcategorized based on diagnostic certainty as definite, probable or possible AMR if 4, 3, or 2 characteristic features were present, respectively. Allograft injury was assessed via plasma donor-derived cell-free DNA (ddcfDNA). Risk of allograft failure and allograft injury was compared for AMR categories using regression models. Over the 38.5 months follow-up, 28.7% of subjects developed clinical AMR (n=96), 18.5% developed subclinical AMR (n=62) or 58.3% were no AMR (n=177). Clinical AMR showed higher risk of allograft failure and ddcfDNA levels compared to subclinical or no AMR. Clinical AMR included definite/probable (n=21) or possible AMR (n=75). These subcategories showed similar clinical characteristics, ddcfDNA levels, and risk of allograft failure. However, definite/probable AMR showed greater measures of AMR severity, including degree of allograft dysfunction and risk of death compared to possible AMR. Clinical AMR showed greater risk of allograft failure than subclinical AMR or no AMR. Subcategorization of clinical AMR based on diagnostic certainty correlated with AMR severity and risk of death, but not with the risk of allograft failure.

Preemptive treatment of de novo donor-specific antibodies in lung transplant patients reduces subsequent risk of chronic lung allograft dysfunction or death

The development of donor-specific antibodies after lung transplantation is associated with downstream acute cellular rejection, antibody-mediated rejection (AMR), chronic lung allograft dysfunction (CLAD), or death. It is unknown whether preemptive (early) treatment of de novo donor-specific antibodies (dnDSAs), in the absence of clinical signs and symptoms of allograft dysfunction, reduces the risk of subsequent CLAD or death. We performed a multicenter, retrospective cohort study to determine if early treatment of dnDSAs in lung transplant patients reduces the risk of the composite endpoint of CLAD or death. In the cohort of 445 patients, 145 patients developed dnDSAs posttransplant. Thirty patients received early targeted treatment for dnDSAs in the absence of clinical signs and symptoms of AMR. Early treatment of dnDSAs was associated with a decreased risk of CLAD or death (hazard ratio, 0.36; 95% confidence interval, 0.17-0.76; P < .01). Deferring treatment until the development of clinical AMR was associated with an increased risk of CLAD or death (hazard ratio, 3.00; 95% confidence interval, 1.46-6.18; P < .01). This study suggests that early, preemptive treatment of donor-specific antibodies in lung transplant patients may reduce the subsequent risk of CLAD or death.

Modelling acute antibody-mediated rejection of human kidney transplants using ex-vivo warm machine perfusion.

Transplant rejection is a major cause of graft loss and morbidity. Currently, no human models of antibody-mediated rejection (AMR) exist, limiting mechanistic investigation and organ-specific targeted therapy. Here, using 12 human kidneys and ex-vivo normothermic machine perfusion, we demonstrate phenotypes of AMR after addition of antibodies against either human HLA class I or blood group antigens (A, B), thus modelling clinical AMR that can follow HLA incompatible (HLAi) or blood group incompatible (ABOi) transplantation. Discarded human kidneys with wide ranging demographics and cold ischaemia times (11-54h) were perfused with red blood cells and fresh frozen plasma (FFP) as a source of complement/coagulation factors. For the HLAi model, 600μg of W6/32 anti-class 1 HLA antibody was added to the circuit (time '0'). For the ABOi model, high titre FFP of the relevant blood group antibody was added. Renal blood flow index (RBFi, mL/min/100 g), C3 desArg, prothrombin fragments 1+2 and histology were determined. Our endpoints included haemodynamic changes, thrombosis, and biopsy proven complement deposition. Compared to control kidneys perfused without anti-donor antibodies, both models demonstrated haemodynamic collapse after antibody perfusion with only the HLAi model showing glomerular C4d deposition. We show that a clinically relevant human kidney model of AMR is feasible, and anticipate that these models, with refinements, could provide a basis to test different strategies to prevent AMR. The Rosetrees and Stonygate Trust, The Royal College of Surgeons of England Fellowship Grant, NIHR Biomedical Research Centre/KCL Early Career Grant, Kidney Research U.K.

Efficacy and safety of the SARS-CoV-2 mRNA vaccine in lung transplant recipients: a possible trigger of rejection.

ObjectiveSolid organ transplant recipients have an increased risk of developing severe coronavirus disease 2019 (COVID-19). Although SARS-CoV-2 mRNA vaccination has been strongly recommended for solid organ transplant recipients, its efficacy and safety have remained unknown.MethodsWe performed an observational prospective cohort study in 18 lung transplant recipients who received two doses of SARS-CoV-2 mRNA vaccine, including BNT162b2 (n = 17) or mRNA-1273 (n = 1), between June and October 2021. The titers of IgG antibodies against the SARS-CoV-2 spike protein (S-IgG) were measured in serum samples collected before the prime dose, three weeks after the prime dose, and four weeks after the booster dose. Reactogenicity and adverse events were evaluated after vaccination.ResultsThere were no recipients with previous SARS-CoV-2 infection prior to vaccination. S-IgG levels were elevated in 2/18 (11.1%) recipients after the prime dose and in 5/18 recipients (27.8%) after the booster dose (31.7 ± 30.6 U/ml). The time from transplantation to vaccination tended to be longer in the seropositive group than the seronegative group [7.5 (3.9–10.2) vs 2.8 (1.9–4.0) years, p = 0.059]. Maintenance dose of mycophenolate mofetil tended to be lower in the seropositive group than in the seronegative group [500 (250–500) vs 1000 (1000–1000) mg/day, p = 0.088]. Regarding the adverse events after vaccination, the development of chronic lung allograft dysfunction (CLAD) or antibody-mediated rejection (AMR) were observed in two seropositive patients.ConclusionsThe antibody response to the SARS-CoV-2 mRNA vaccine was quite poor in lung transplant recipients. We experienced cases that developed clinical CLAD or AMR that was likely related to SARS-CoV-2 vaccination.

Circulating microRNAs in cellular and antibody-mediated heart transplant rejection.

Noninvasive monitoring of heart allograft health is important to improve clinical outcomes. MicroRNAs (miRs) are promising biomarkers of cardiovascular disease and limited studies suggest they can be used to noninvasively diagnose acute heart transplant rejection. The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter prospective cohort study that phenotyped heart transplant patients from 5 mid-Atlantic centers. Patients who had no history of rejection after transplant were compared to patients with acute cellular rejection (ACR) or antibody-mediated rejection (AMR). Small RNA sequencing was performed on plasma samples collected at the time of an endomyocardial biopsy. Differential miR expression was performed with adjustment for clinical covariates. Regression was used to develop miR panels with high diagnostic accuracy for ACR and AMR. These panels were then validated in independent samples from GRAfT and Stanford University. Receiver operating characteristic curves were generated and area under the curve (AUC) statistics calculated. Distinct ACR and AMR clinical scores were developed to translate miR expression data for clinical use. The GRAfT cohort had a median age of 52 years, with 35% females and 45% Black patients. Between GRAfT and Stanford, we included 157 heart transplant patients: 108 controls and 49 with rejection (50 ACR and 38 AMR episodes). After differential miR expression and regression analysis, we identified 12 miRs that accurately discriminate ACR and 17 miRs in AMR. Independent validation of the miR panels within GRAfT led to an ACR AUC 0.92 (95% confidence interval [CI]: 0.86-0.98) and AMR AUC 0.82 (95% CI: 0.74-0.90). The externally validated ACR AUC was 0.72 (95% CI: 0.59-0.82). We developed distinct ACR and AMR miR clinical scores (range 0-100), a score ≥ 65, identified ACR with 86% sensitivity, 76% specificity, and 98% negative predictive value, for AMR score performance was 82%, 84% and 97%, respectively. We identified novel miRs that had excellent performance to noninvasively diagnose acute rejection after heart transplantation. Once rigorously validated, the unique clinical ACR and AMR scores usher in an era whereby genomic biomarkers can be used to screen and diagnose the subtype of rejection. These novel biomarkers may potentially alleviate the need for an endomyocardial biopsy while facilitating the initiation of targeted therapy based on the noninvasive diagnosis of ACR or AMR.

Eight-Year Results of an IgA- and IgM-Enriched Human Immunoglobulin-Based Therapy for Early Detectable Anti-HLA Donor Specific Antibodies After Lung Transplantation

<h3>Purpose</h3> The development of anti-HLA donor specific antibodies early after lung transplantation (eDSA) has been associated with antibody-mediated rejection (AMR) and poor graft survival. At our institution, since 2013, successive infusions of IgA- and IgM-enriched human intravenous immunoglobulins (IgGAM, first infusion: 2gr/kg, then 0.5gr/kg once every 4 weeks for a maximum of 6 months) formed the backbone of eDSA therapy. Actually, patients with only evidence of eDSA (possible subclinical AMR) are treated with IgGAM only. Patients with concomitant graft dysfunction (possible clinical AMR) or preformed DSA (positive virtual crossmatch) receive additionally plasmapheresis (PE) or immunoabsorption before the first IgGAM dose, and a single dose of anti-CD20 antibody (Rituximab) thereafter. Aims of this study were to present the 8-year results of the IgGAM-based therapy. <h3>Methods</h3> Records of patients transplanted between 02/2013 and 10/2021 were reviewed. Outcomes were compared between patients with eDSA and treated with IgGAM (IgGAM group) and without eDSA (control group)<i>.</i> Median (IQR) follow-up was 45 (21-72) months. <h3>Results</h3> During the study period, among the 994 transplanted patients, 250 (25%) patients formed the IgGAM group and 721 (73%) the control group. The remaining 23 (2%) patients (14 patients with eDSA but not treated, and 9 patients treated only with tPE and Rituximab) were excluded. Fifty-Five IgGAM patients (22%) showed pre-formed eDSA and 41 (16%) patients showed possible clinical AMR. Median time to eDSA detection was 14 days after transplantation. At follow-up end, treatment was completed in 229 (92%) patients (still on treatment, n=8; in-hospital deaths, n=4; treatment interrupted earlier as intended by protocol, n=9). In these patients, IgGAM treatment cleared eDSA in 208 (91%) patients, 29 (14%) patients showing eDSA recurrence a median of 12 months after treatment end. In IgGAM vs. control patients and at 5- and 8-year follow-up, respectively, graft survival (%) was 77 vs. 76 and 68 vs. 67 (p=0.53), and freedom from CLAD was 73 vs. 72 and 65 vs. 62 (p=0.76), respectively. <h3>Conclusion</h3> After lung transplantation, a treatment protocol for eDSA based on IgGAM yielded high eDSA clearance. Patients with eDSA and IgGAM-treatment have good 8-year graft survival similar to control patients.

Anti-HLA DQ Antibodies Are Associated with Chronic Lung Allograft Dysfunction in a Pediatric Lung Transplant Population

<h3>Purpose</h3> Chronic lung allograft dysfunction (CLAD) limits long term survival after lung transplantation. Antibody mediated rejection (AMR) has been associated with earlier CLAD development, but studies are limited in children. Clinical AMR can exist on a spectrum from subclinical to definite AMR based on the presence of anti-HLA donor specific antibodies (DSA), allograft dysfunction, and histopathologic findings, with DSA development first to appear. This study aims to describe the relationship of elevated DSA with clinical outcomes in a single center pediatric lung transplant population. <h3>Methods</h3> This was a single center, retrospective study. Pediatric bilateral lung transplant recipients were included from 2002-2020 if they had longitudinal HLA and lung function data for at least 6 months after transplantation. Preliminary analyses with chi-squared or Fisher's exact test for categorical variables and ANOVA or Kruskal-Wallis for continuous variables were used. DSA were detected with single-antigen bead arrays, and elevated DSA was defined as a mean fluorescence intensity level above 500. <h3>Results</h3> 34 patients were included: 19 with elevated DSA and 15 without. Most patients had subclinical AMR (n=10) without allograft dysfunction and two patients had definite AMR, based on the 2018 Banff consensus statement. The proportion of patients who developed CLAD was significantly higher in the elevated DSA group and had an earlier onset of CLAD. Average graft longevity and rates of acute cellular rejection, while not significant, trended towards shorter survival and higher rates of rejection in the elevated DSA group. All persistently elevated DSA were directed against HLA Class II DQ (6 of 14 patients) epitopes. Survival analysis and the significance of different HLA antigens remains ongoing. <h3>Conclusion</h3> The development of persistently elevated HLA Class II DQ DSA is associated with CLAD and likely decreased survival in the pediatric lung transplant population.

First Report of Daratumumab in Clinical Lung Transplantation

<h3>Purpose</h3> Antibody mediated rejection (AMR) after lung transplantation is difficult to treat and often results in death or graft loss. Therapies targeting antibodies or B cells are in many cases inadequate for decreasing donor specific antibodies (DSA), particularly when they are directed against MHC class II. Daratumumab (DAR) is a humanized anti-CD38 monoclonal antibody, which induces plasma cell death through multiple mechanisms including complement- dependent cytotoxicity, antibody-dependent phagocytosis, and apoptosis. Based on these properties it may have the potential to reduce the amount of DSAs and therefore improve outcome after AMR. <h3>Methods</h3> Retrospective analysis of all patients who received daratumumab as an add on rescue therapy for AMR or for desensitization pre/post-transplant at our center. <h3>Results</h3> Six patients received daratumumab due to the following reasons: 5 patients with de novo DSAs and AMR, 1 patient with pre-transplant DSAs and post-transplant immunadsorption without clinical AMR. Daratumumab was safely administered with just mild infusion reactions and no severe adverse event. Of not, none of the patients developed and infectious complication. Five of the 6 patients showed a significant decrease of their DSAs with a reduction of MFI values after 6-8 weeks to <50% of the baseline . Despite this early success, four patients developed CLAD (,two of which required retransplantation. The remaining two patients stabilized with their lung function and did not develop CLAD. <h3>Conclusion</h3> Treatment for AMR remains challenging, especially in the presence of class II HLA antibodies. Daratumumab might be a promising addition to the AMR treatment panel, prospective clinical studies are needed.

Treatment of De Novo DSA with IVIG Monotherapy After Lung Transplantation

<h3>Purpose</h3> The development of de novo donor-specific anti-HLA antibodies (DSA) is associated with worse outcomes in lung transplant recipients (LTxR). The purpose of this study is to evaluate IVIG monotherapy for de novo DSA in the absence of allograft dysfunction or clinical antibody-mediated rejection (AMR). <h3>Methods</h3> Adult LTxR between 2/2013 and 3/2021 treated with IVIG monotherapy for de novo DSA with no evidence of AMR were included. IVIG was initiated at 2g/kg followed by 1g/kg monthly for a minimum of 3 months or until clearance (up to 6 months). HLA Ab is routinely checked by Luminex SAB post-transplant at day 14, months 1, 3, 6, 9, 12, and 18, and monthly during IVIG treatment. Primary outcome was change in DSA after IVIG therapy. DSA clearance was defined as sum MFI less than 1000. <h3>Results</h3> 32 LTxR developed de novo DSA at a median of 33 days (18,143) post-transplant and were treated with IVIG. Mean follow-up time was 1330 days (861-1910). The median sum MFI for DSA at baseline was 4782 (2927,7490). DQ was the immunodominant DSA in 24 (75%) and only 2 (6%) patients had isolated class I DSA. MFI of sum DSA decreased by a median of 2993 (2051, 6358) after IVIG therapy; p < 0.0001. Eighteen (56%) cleared de novo DSA (Group 1) versus 14 (44%) did not clear (Group 2) at the end of IVIG therapy. There was no significant difference in age, gender, race, transplant indication or type, cPRA, HLA mismatch, or PGD. ACR and AMR tended to be more frequent in Group 2; ACR: 92.9% vs 61.1%, p=0.053; and AMR: 35.7% vs 5.6%, p=0.063. Overall survival for the entire cohort at 1, 3 and 5 years was 100%, 86%, 60%, respectively. There was no significant difference in chronic lung allograft dysfunction (CLAD) or survival between Groups (Table 1). <h3>Conclusion</h3> In LTxR, IVIG monotherapy is associated with significant reduction in de novo DSA. DSA clearance may result in less acute rejection. Randomized controlled trials are needed to determine the optimal strategy for managing de novo DSA.

Circulating microRNA Biomarkers in Cellular and Antibody-Mediated Heart Transplant Rejection

<h3>Purpose</h3> Non-invasive monitoring of heart allograft health is an important clinical goal and circulating microRNAs (miRs) have been associated with acute rejection in limited studies. <h3>Methods</h3> The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter prospective study. Patients with no history of rejection after transplant were selected as controls. Cases were patients with acute cellular rejection (ACR) or antibody-mediated rejection (AMR). Small RNA sequencing was performed on plasma samples collected at the time of an endomyocardial biopsy. Differential miR expression analysis and LASSO regression was used to develop ACR and AMR miR panels while adjusting for clinical covariates. The panels were validated in an independent sample from GRAfT and Stanford University. Receiver operating characteristic curves were generated and area under the curve (AUC) statistics are reported. Distinct ACR and AMR clinical scores were developed to translate gene expression data for clinical use. <h3>Results</h3> The GRAfT cohort had a median age of 52 years, with 35% females and 45% Blacks. We included 85 controls and 34 patients with rejection (20 ACR, 14 AMR). In ACR and AMR, using differential gene expression and regression analysis, we identified 11 and 15 miRs that accurately discriminated ACR and AMR (p<0.05). Independent validation of the miR panels within GRAfT led to excellent diagnostic accuracy for ACR AUC 0.92 (95% CI: 0.86-0.98) and AMR AUC 0.82 (95% CI: 0.74-0.90). The externally validated ACR AUC was 0.91 (95% CI: 0.82-0.99). Distinct ACR and AMR miR clinical scores were developed ranging from 0-100; using a threshold of 65 identified ACR with a sensitivity of 86%, specificity of 78% and negative predictive value of 98%, for AMR the respective performance was 82%, 84% and 97% (Figure). <h3>Conclusion</h3> The unique clinical ACR and AMR scores usher in an era whereby genomic biomarkers are used not only for rejection surveillance but permit accurate, non-invasive diagnosis of ACR and AMR.

Contrary to Persisting De-Novo DSA, Transient De-Novo DSAs After Lung Transplantation Do Not Constitute a Risk Factor for Patient and Graft Survival

<h3>Purpose</h3> Approximately 50% of lung transplantation (LuTx) recipients develop de-novo DSA (dnDSAs) within the first year. In our institution, therapy is started only in case of clinical antibody-mediated rejection. The majority of dnDSAs disappears within 3-6 months; however, in some patients they persist. The aim of the study was to determine whether persistence of dnDSAs is associated with worse outcomes and as a consequence is worth of therapeutic intervention <h3>Methods</h3> All patients, transplanted between 2010 and 2020. dnDSAs were defined as persistent if detectable for a time period longer than 6 months, otherwise were classified as transient. Four categories of mean fluorescence intensity were defined: I (1000-5000), II (5000-10000), III (10000-15000) and IV (>15000). <h3>Results</h3> 730 patients were included in the analysis, 153 patients (21%) developed dnDSAs. Eighty-one (11%) were transient while in 71 patients (10%) persisted for a period longer than six months. MFI categories were significantly different between transient and persistent dnDSAs (p<0.001): I (88% vs 17%), II (7% vs 33%), III (5% vs 19%), IV (0% vs 31%). 3 patients (4%) in the transient group and 26 (36%) in the persistent group developed clinical AMR (p<0.001) during the study period. Freedom from CLAD at 1-, 3- and 5-years was 93%, 86% and 63% in transient dnDSAs group compared to 85%, 49% and 34% in persistent dnDSAs group (p=0.001). Graft survival at 1-, 3- and 5-years was 90%, 78% and 68% in transient dnDSAs group compared to 81%, 59% and 45% in persistent dnDSAs group (p=0.008). Of not, the transient dnDSAs group showed similar 5-year survival as patients without dnDSAs (69% vs 74%, p=0.833). Contrarily, patients with persistent dnDSAs had low 5-year survival of only 48% (p=0.002). <h3>Conclusion</h3> Persistent but not transient dnDSAs are associated with worse long-term outcomes. Further studies are needed to evaluate possible therapeutic strategies, aimed to prevent subclinical damage to the allograft.

Lung Transplant Recipients Developing Early DSAs within the First Month Are Characterized by a Higher Frequency of Naïve and a Lower Frequency of Memory B Cells

<h3>Purpose</h3> After lung transplantation (LuTx), the development of early donor-HLA-specific antibodies (eDSA) has been shown to be associated with antibody-mediated rejection (AMR) and poor graft survival. Since 2013, patients with eDSA within the first month after LuTx are treated with IgA/IgM-enriched intravenous immunoglobulins (IgGAM), in clinical AMR combined with anti-CD20 antibody (Rituximab). We hypothesize a different distribution and kinetics of IgD⁺CD27^- naïve B cells vs. IgD^-CD27^+/- memory B cell subsets in recipients with or without eDSA. <h3>Methods</h3> In a pilot study of 62 LuTx recipients, B cell subsets were by analyzed in blood pre, post (T0), 24 hours (T24) and 3 weeks after LuTx by flow cytometry using CD19, CD20, IgD, CD24, CD27, CD38 antibodies. Frequencies of B cell subsets were compared between two groups, eDSA-positive (n=14, 22,6%) and -negative (n=48, 77,4%) patients and healthy donors (n=30). <h3>Results</h3> Prior to LuTx, patients with end-stage lung disease showed significantly higher proprotions of IgD⁺CD27^- naïve B cells compared to HC (p<0.001). At T0, T1 and T24, patients with eDSAs showed significantly higher frequencies of naïve and lower frequencies of IgD^-CD27⁺ memory B cells (both p< 0.001) compared to eDSA-negative patients. These IgD⁺CD27^- naïve B cells displayed high CD24 and CD38 expression, which was stable also at 3 wks. A transient increase in IgD⁺CD27⁺ switch memory B cells was detected at T0 in both groups, returning to baseline levels at T24. <h3>Conclusion</h3> Lung transplant recipients show remarkable dynamics of naïve, memory and switch memory B cells within the first 3 wks. In both groups, we saw a shift in the B cell population: increased naïve B cells and decreased memory B cells, which was more pronounced in the eDSA group. Based on our preliminary data, a more refined B cell monitoring with additional markers may be able to identify patients with a higher risk for eDSA development. The impact of the treatment regimen on these B cell subsets is currently investigated.

Clinical Relevance of Isoagglutinin Rebound in Adult ABO-Incompatible Living Donor Liver Transplantation.

ABO-incompatible (ABO-I) living donor liver transplantation (LDLT) can be performed successfully. However, anti-ABO isoagglutinin rebound may cause antibody-mediated rejection (AMR) and graft loss. The risk threshold of isoagglutinin rebound is still not defined. 76 ABO-I LDLT recipients were divided into group A (n = 56) with low isoagglutinin titers (<1:256), and group B (n = 20) with high isoagglutinin titers (≥1:256), at initial assessment for liver transplantation. The last 12 patients in group B received a modified desensitization regimen by adding bortezomib to deplete plasma cells. Six (10.7%) patients in group A and 10 (50.0%) patients in group B had postoperative isoagglutinin rebound (p < 0.001). Three patients (5.54%) in group A and two patients (10%) in group B developed clinical AMR (p = 0.602). The cutoff value of postoperative isoagglutinin rebound to cause clinical AMR was ≥1:1024. Among the 12 patients in group B with bortezomib administration, isoagglutinin rebounded up to 1:128 only, and no clinical AMR occurred. In conclusion, the patients with high isoagglutinin titers had a higher rate of postoperative isoagglutinin rebound. Isoagglutinin rebound ≥1:1024 is risky for developing clinical AMR. Adding bortezomib into the desensitization regimen may mitigate isoagglutinin rebound, and avoid clinical AMR.

Outcome of Husband-to-Wife Kidney Transplantation With Mutual Children: Single Center Experience Using T Cell-Depleting Induction and Review of the Literature.

Few data on husband-to-wife transplantations with mutual children (H2W) exist in the current era. We investigated the outcome of H2W transplantations (n = 25) treated with T cell-depleting induction compared to women with prior pregnancies also receiving their first HLA-mismatched kidney transplant, but from a different donor source: (i) other living donor (n = 52) and (ii) deceased donor (n = 120). Seventy-four percent of the women had ≥2 pregnancies; median follow-up time was 5 years. Death-censored allograft survival was significantly lower in the H2W group compared to the other two groups (p = 0.03). Three of four graft losses in the H2W group were due to rejection. 5-year patient survival in the H2W group was high and similar compared to the other living donor group (100 vs. 98%; p = 0.28). The incidence of (sub)clinical antibody-mediated rejection was higher in the H2W group (36 vs. 20 vs. 18%) (p = 0.10). The frequency of infections was similar among the three groups. No immunological parameter was predictive for rejection or graft loss in H2W transplantations. In conclusion, H2W transplantation is a valuable option, but associated with a higher risk for allograft loss due to rejection despite T cell-depleting induction. Further research is required for better risk prediction on an individual patient level.

Seven-Year Clinical Results of an IgA-and IgM-Enriched Human Immunoglobulin-Based Therapy for Antibody-Mediated Rejection after Lung Transplantation

Purpose Development of anti-HLA donor specific antibodies early after lung transplantation (eDSA) has been associated with antibody-mediated rejection (AMR) and poor graft survival. Since 2013, our institution has employed successive infusions of IgA- and IgM-enriched intravenous immunoglobulins (IgGAM, first infusion: 2gr/kg, then 0.5gr/kg once every 4 weeks for a maximum of 6 months) as 1st line treatment in such cases. Asymptomatic eDSA development (possible subclinical AMR) was treated with IgGAM only. Patients with concomitant graft dysfunction (possible clinical AMR) or positive crossmatch additionally receive 5 sessions of plasmapheresis pre-IgGAM, and a single dose of anti-CD20 antibody (Rituximab) afterwards. This study was presents 7-year results of this IgGAM-based protocol. Methods Records of patients transplanted between 02/2013 and 10/2020 were reviewed. Outcomes were compared between patients with eDSA and treated with IgGAM (IgGAM group) and those without eDSA (control group). Median follow-up was 41 (18-64) months. Results he 913 transplanted recipients, 224 (25%) patients were included in the IgGAM group and 667 (73%) the control group. The remaining 22 (2%) patients (13 patients with eDSA but not treated, and 9 patients treated only with tPE and Rituximab) were excluded. Forty-one (18%) IgGAM patients exhibited pre-formed eDSA and 16 (7%) patients showed a positive retrospective crossmatch. Thirty-six (16%) patients showed concomitant graft dysfunction (possible clinical AMR). Median time to eDSA detection was 14 days post-transplant. At follow-up end, treatment was completed in 204 (91%) patients (ongoing treatment, n=7; in-hospital deaths, n=4; treatment prematurely interrupted, n=9). In these 204 patients, IgGAM treatment cleared eDSA in 184 (90%) patients. Twenty-five (12%) patients developed eDSA recurrence at a median of 9 months after completing treatment. Clearance was less effective in patients with preformed eDSA (p Conclusion In lung recipients, treatment of eDSA with IgGAM yielded high eDSA clearance. Patients with eDSA and IgGAM-treatment have good 7-year graft survival similar to control patients.

Humoral Risk Factors Associated to Allograft Dysfunction after Lung Transplantation: The Alert of Non-HLA Auto Antibody and HLA-Donor Specific Antibody (DSA) with Non-DSA HLA Antibody

<h3>Purpose</h3> Growing evidence has indicated the negative impact of non-HLA auto antibody (NHAuAb) on organ transplantation. Donor specific antibody (DSA) after lung transplantation (LuT) has been observed frequently with de novo Non-DSA (dN-DSA) HLA antibody. This study investigates the deleterious role of post-LuT NHAuAb and dN-DSA synergism to DSA in allograft damage. <h3>Methods</h3> We retrospectively analyzed NHAuAb and de novo DSA (dDSA)/dN-DSA data collected from the recipients who underwent LuT during 2012-2016. NHAuAbs were detected using LABScreen™ Autoantibody (>95% non-sensitized subjects as cut-off). HLA antibody was tested by LABScreen™ SAB (MFI≥ 1000 as cut-off). Clinical Antibody Mediated Rejection (cAMR, with acute graft dysfunction) and Chronic Lung Allograft Dysfunction (CLAD) were assessed for LuT outcomes. <h3>Results</h3> CXCL9, AURKA, REG3A, IFNγ, FLRT2, GDNF, HNRNPK, CXCL10, PLA2R, and Collagen IV detected from 111 patients' sera drawn at DSA peak phase showed significant association to total AMR, but only FLRT2 (P<0.001), GDNF, and CXCL10 (P<0.05) were remarkably correlated to cAMR (Table 1a). Preformed & persistent LG3, PECR, PKC-Z (P<0.001) and de novo ENO1, GAPDH, Lamin-B, TubulinA1B (p<0.05) identified from 113 patients' sera drawn around CLAD time, demonstrated strong correlation to the occurrence of CLAD (Table 1b). Patients with dDSA & dN-DSA had significantly higher incidence of cAMR than the patients with only dDSA (p<0.001, Table 1c). <h3>Conclusion</h3> Distinct clinical phases presented specific NHAuAb profiles. Multiple NHAuAbs, especially LG3, PKC-Z, and PECR which were preformed & persistent, promoted chronic graft dysfunction. HLA-dDSA augmented by dN-DSA (most likely through shared targeting epitopes) and NHAuAbs of FLRT2, GDNF, and CXCL10 were related to acute graft injury. These HLA and non-HLA humoral risk factors warrant timely enhanced immune therapy to prevent final allograft failure.