Donor-specific HLA Antibodies Research Articles

Pre-Transplant Angiotensin II Type 1 Receptor Antibodies and Anti-Endothelial Cell Antibodies Predict Graft Function and Allograft Rejection in a Low-Risk Kidney Transplantation Setting.

BackgroundNon-HLA antibodies, anti-angiotensin II type 1 receptor antibodies (anti-AT1R) and anti-endothelial cell antibodies (AECA), are known to play a role in allograft rejection. We evaluated the role of both antibodies in predicting post-transplant outcomes in low-risk living donor kidney transplantation (LDKT) recipients.MethodsIn 94 consecutive LDKT recipients who were ABO compatible and negative for pre-transplant HLA donor-specific antibodies, we determined the levels of anti-AT1Rs using an enzyme-linked immunosorbent assay and the presence of AECAs using a flow cytometric endothelial cell crossmatch (ECXM) assay with pre-transplant sera. Hazard ratio (HR) was calculated to predict post-transplant outcomes.ResultsPre-transplant anti-AT1Rs (≥11.5 U/mL) and AECAs were observed in 36 (38.3%) and 22 recipients (23.4%), respectively; 11 recipients had both. Pre-transplant anti-AT1Rs were a significant risk factor for the development of acute rejection (AR) (HR 2.09; P=0.018), while a positive AECA status was associated with AR or microvascular inflammation only (HR 2.47; P=0.004) throughout the follow-up period. In particular, AECA (+) recipients with ≥11.5 U/mL anti-AT1Rs exhibited a significant effect on creatinine and estimated glomerular filtration rate (P<0.001; P=0.028), although the risk of AR was not significant.ConclusionsPre-transplant anti-AT1Rs and AECAs have independent negative effects on post-transplant outcomes in low-risk LDKT recipients. Assessment of both antibodies would be helpful in stratifying the pre-transplant immunological risk, even in low-risk LDKT recipients.

CLINICAL SIGNIFICANCE OF PRETRANSPLANT DONOR-SPECIFIC HLA ANTIBODIES IN KIDNEY TRANSPLANT RECIPIENTS OF HISPANIC POPULATION

Introduction: Donor-specific HLA antibodies (DSA) before kidney transplantation (KT) is one of the major risk factors for humoral rejection and lower graft survival (1-2). In order to identify prognostic factors and develop follow up strategies in this high risk population, it is required for transplant center to share their outcome data (3-4). In addition, in most previous studies the Hispanic population is poorly represented. Materials and Methods: Retrospective cohort and comparative study in KT recipients with pretransplant DSA measured by Luminex, negative AHG-CDC-XM (living and deceased donor) and negative flow cross match (living donor). Our main objective was to determine graft survival and describe the incidence of acute rejection (AR) and renal function (eGFR by CKD-EPI). Results: Between September 2006 and March 2017, 411 patients received KT in the INCMNSZ, 60 were identified with pretransplant DSA and compared with 60 KT recipients without pretransplant DSA, paired by donor type, induction and maintenance immunosuppression. Median age was 33 (range 27-44) years, 55 (45%) were women, 55 (45.8%) had unknown etiology of ESRD, months on dialysis [24 (range 12-48)], 10.7% received second KT, 63.3% were living donor receptor, 75% had thymoglobulin induction, 96% were with tacrolimus, mycofenolate mofetil and steroids as maintenance immunosupression. The incidence of AR was increased in the pretransplant DSA group (35.5% vs. 15.2%, p=0.011) and the median time between KT and AR episodes was lower in the pretransplant DSA group [12.8 (8.3-23.6) vs 32.1 (25.9-40.6), (p<0.0001)] months.In the pretransplant DSA group 52.4% of the biopsies with AR were protocol biopsies and 29% were secondary to graft disfunction.Incidence of De Novo DSA were similar between groups [30 (53.6%) vs 29 (56.8%), (p=0.732)].After 37.4 (range 29.2-52.3) months of follow up, eGFR was similar between groups 65.0 ± 22.0 vs 69.8 ± 21.4 ml/min/1.73m2 (p=0.19) and there were no difference in graft survival (87.7% vs 96.7% p=0.240) between pretransplant DSA and without pretransplant DSA groups, respectively.Discussion and conclusions: Although there is a higher incidence of rejection, KT recipients with pretransplant DSA could be considered candidates for KT. One limitation of this study is the small sample size and short follow-up time. However, we acknowledge that in this population of high immunological risk, induction therapy with lymphocyte depletors and powerful maintenance immunosuppression, combined with follow-up strategies as protocol biopsies with early treatment of subclinical rejection could provide similar graft survival compared to KT receptors without pretransplant DSA.

PRE-TRANSPLANT HLA ANTIBODIES AND DELAYED GRAFT FUNCTION IN THE CURRENT ERA OF KIDNEY TRANSPLANTATION

Collaborative Transplant Study. Introduction: We reported previously that early adverse events in deceased donor kidney transplantation, such as delayed graft function (DGF) and rejection episodes, are associated with pre-transplant presence of HLA antibodies and that patients with adverse events show significantly impaired graft survival rates. In the meantime, the introduction of sensitive antibody detection techniques is expected to have diminished the involvement of overlooked HLA antibodies in DGF. On the other hand, DGF risk might have increased due to growing use of kidneys from elderly donors. The aim of the present multi-center study was to identify the immunological and non-immunological predictors of DGF and to determine its influence on outcome in the presence and absence of HLA antibodies in the new era of transplantation. Methods: 1,724 patients who received a deceased donor kidney transplant during 2008–2017 and on whom a pre-transplant serum sample and the information on immediate function within the first 24 hours after transplantation, dialysis during the first week, and biopsy-proven rejection during the first 3 months were available. Graft survival after day 7 during the first 3 years post-transplant was analyzed by multivariable Cox regression. Predictors of DGF and influence of DGF and pre-transplant HLA antibodies on biopsy-proven rejections during days 8–90 were determined by multivariable logistic regression. DGF was defined as either no graft function during the first 24 hours and/or dialysis during the first week. In 44% of cases, we were informed by the centers on the presence or absence of pre-transplant donor-specific antibodies (DSA) as determined by single antigen technique. Results and Discussion: Donor age ≥50 years, simultaneous presence of HLA class I and II antibodies pre-transplant, diabetes mellitus as original disease, cold ischemia time ≥18 hours, and time on dialysis >5 years were associated with an increased risk of DGF. DGF alone doubled the risk for all cause graft loss, more due to impaired death-censored graft than patient survival. In DGF patients, the risk of death-censored graft loss increased further if HLA antibodies (HR=4.75, P<0.001) or donor-specific HLA antibodies (DSA, HR=7.39, P<0.001) were present prior to transplantation. In the presence of HLA antibodies or DSA, the incidence of biopsy-proven rejections increased significantly in patients with as well as without DGF. Recipients without DGF and without biopsy-proven rejections during the first 3 months had the highest fraction of patients with good kidney function at year 1 post-transplant, whereas patients with both DGF and rejection showed the lowest rate of good kidney function. Our data indicate that, in the current era of transplantation, besides non-immunological factors, also the pre-transplant presence of HLA class I and II antibodies increase the risk of DGF. Conclusion: Patients with DGF require special attention, especially in the presence of HLA antibodies.

Pre-transplant HLA Antibodies and Delayed Graft Function in the Current Era of Kidney Transplantation.

Delayed graft function (DGF) occurs in a significant proportion of deceased donor kidney transplant recipients and was associated with graft injury and inferior clinical outcome. The aim of the present multi-center study was to identify the immunological and non-immunological predictors of DGF and to determine its influence on outcome in the presence and absence of human leukocyte antigen (HLA) antibodies. 1,724 patients who received a deceased donor kidney transplant during 2008–2017 and on whom a pre-transplant serum sample was available were studied. Graft survival during the first 3 post-transplant years was analyzed by multivariable Cox regression. Pre-transplant predictors of DGF and influence of DGF and pre-transplant HLA antibodies on biopsy-proven rejections in the first 3 post-transplant months were determined by multivariable logistic regression. Donor age ≥50 years, simultaneous pre-transplant presence of HLA class I and II antibodies, diabetes mellitus as cause of end-stage renal disease, cold ischemia time ≥18 h, and time on dialysis >5 years were associated with increased risk of DGF, while the risk was reduced if gender of donor or recipient was female or the reason for death of donor was trauma. DGF alone doubled the risk for graft loss, more due to impaired death-censored graft than patient survival. In DGF patients, the risk of death-censored graft loss increased further if HLA antibodies (hazard ratio HR=4.75, P < 0.001) or donor-specific HLA antibodies (DSA, HR=7.39, P < 0.001) were present pre-transplant. In the presence of HLA antibodies or DSA, the incidence of biopsy-proven rejections, including antibody-mediated rejections, increased significantly in patients with as well as without DGF. Recipients without DGF and without biopsy-proven rejections during the first 3 months had the highest fraction of patients with good kidney function at year 1, whereas patients with both DGF and rejection showed the lowest rate of good kidney function, especially when organs from ≥65-year-old donors were used. In this new era of transplantation, besides non-immunological factors, also the pre-transplant presence of HLA class I and II antibodies increase the risk of DGF. Measures to prevent the strong negative impact of DGF on outcome are necessary, especially during organ allocation for presensitized patients.

Autoantibodies and Donor-specific Antibodies are Associated With Graft Dysfunction in Pediatric Liver Transplantation.

Autoantibodies (AAb) and donor-specific HLA antibodies (DSA) are frequently present in pediatric liver transplant (LT) recipients. Their clinical significance remains incompletely understood. We aimed to investigate the prevalence of serum AAb and DSA in pediatric LT recipients and its correlation with patient characteristics and histological and biochemical parameters. We retrospectively reviewed the data from 62 pediatric LT patients in follow-up at Ghent University Hospital between January 2007 and February 2018. Blood samples with AAb measurement were taken systematically, liver biopsies (LB) were performed on clinical indication. AAb were detected in 27 (43.3%) patients, with antinuclear antibodies (ANA) being the most frequently (24%) encountered AAb. There was an association between AAb positivity and female gender (P = 0,032) and deceased donor LT (P = 0,006). Patients with positive AAb underwent a higher number of LB during their follow-up (P < 0,001), and an association was found with the presence of nonspecific histologic alterations (P = 0,032) in the absence of de novo autoimmune hepatitis. Positive AAb were also associated with higher alkaline phosphatase (P < 0,001), ALT (P < 0,001), AST (P < 0,001), γ-GT (P = 0,001), IgG (P = 0,011) and lower albumin (P = 0,029). Fourteen out of 50 (28%) patients were DSA-positive, mostly anti-HLA class II. DSA positivity was associated with T-cell-mediated rejection (P = 0,019), higher total (P = 0,033), and direct (P = 0,012) bilirubin and γ-GT (P < 0,001). The presence of AAb and DSA is associated with histological and biochemical parameters of graft dysfunction. Larger prospective studies are warranted to investigate the causal relationships between AAb and DSA development and outcome parameters post pediatric LT.

The Histological Picture of Indication Biopsies in the First 2 Weeks after Kidney Transplantation.

In preclinical studies, ischemia-reperfusion injury and older donor age are associated with graft inflammation in the early phase after transplantation. In human kidney transplantation, impaired allograft function in the first days after transplantation is often adjudicated to donor- and procedure-related characteristics, such as donor age, donor type, and ischemia times. , setting, participants, & measurementsIn a cohort of 984 kidney recipients, 329 indication biopsies were performed within the first 14 days after transplantation. The histologic picture of these biopsies and its relationship with alloimmune risk factors and donor- and procedure-related characteristics were studied, as well as the association with graft failure. Multivariable Cox models were applied to quantify the cause-specific hazard ratios for early rejection and early inflammatory scores, adjusted for potential confounders. For quantification of hazard ratios of early events for death-censored graft failure, landmark analyses starting from day 15 were used. Early indication biopsy specimens displayed microvascular inflammation score ≥2 in 30% and tubulointerstitial inflammation score ≥2 in 49%. Rejection was diagnosed in 186 of 329 (57%) biopsies and associated with the presence of pretransplant donor-specific HLA antibodies and the number of HLA mismatches, but not nonimmune risk factors in multivariable Cox proportional hazards analysis. In multivariable Cox proportional hazards analysis, delayed graft function, the graft dysfunction that prompted an early indication biopsy, HLA mismatches, and pretransplant donor-specific HLA antibodies were significantly associated with a higher risk for death-censored graft failure, whereas early acute rejection was not. Indication biopsies performed early after kidney transplantation display inflammatory changes related to alloimmune risk factors. Nonimmune risk factors for ischemia-reperfusion injury, such as cold and warm ischemia time, older donor age, and donor type, were not identified as strong risk factors for early inflammation after human kidney transplantation.

Eplet Mismatch Load and De Novo Occurrence of Donor-Specific Anti-HLA Antibodies, Rejection, and Graft Failure after Kidney Transplantation: An Observational Cohort Study.

In kidney transplantation, evaluating mismatches of HLA eplets-small patches of surface-exposed amino acids of the HLA molecule-instead of antigen mismatches might offer a better approach to assessing donor-recipient HLA incompatibility and improve risk assessment and prediction of transplant outcomes. To evaluate the effect of number of eplet mismatches (mismatch load) on de novo formation of donor-specific HLA antibodies (DSAs) and transplant outcomes, we conducted a cohort study that included consecutive adult kidney recipients transplanted at a single center from March 2004 to February 2013. We performed retrospective high-resolution genotyping of HLA loci of 926 transplant pairs and used the HLAMatchmaker computer algorithm to count HLA eplet mismatches. De novo DSAs occurred in 43 (4.6%) patients. Multivariable analysis showed a significant independent association between antibody-verified eplet mismatch load and de novo DSA occurrence and graft failure, mainly explained by DQ antibody-verified eplet effects. The association with DQ antibody-verified eplet mismatches was linear, without a safe threshold at which de novo DSA did not occur. Odds for T cell- or antibody-mediated rejection increased by 5% and 12%, respectively, per antibody-verified DQ eplet mismatch. Eplet mismatches in HLA-DQ confer substantial risk for de novo DSA formation, graft rejection, and graft failure after kidney transplantation. Mismatches in other loci seem to have less effect. The results suggest that antibody-verified HLA-DQ eplet mismatch load could be used to guide personalized post-transplant immunosuppression. Adoption of molecular matching for DQA1 and DQB1 alleles could also help to minimize de novo DSA formation and potentially improve transplant outcomes.

Guidelines for Cord Blood Unit Selection.

Optimal cord blood (CB) unit selection is critical to maximize the likelihood of successful engraftment and survival after CB transplantation (CBT). However, unit selection can be complex because multiple characteristics must be considered including unit cell dose, donor-recipient human leukocyte antigen (HLA) match, and unit quality. This review provides evidence-based and experience-based comprehensive guidelines for CB unit selection. Topics addressed include the use of both the TNC and the CD34+ cell dose, as well as the CD34+ cell to TNC content ratio to evaluate unit progenitor cell content and engraftment potential, the acceptable TNC and CD34+ cell dose criteria that define an adequate single-unit graft, and the indication and acceptable cell dose criteria for double-unit grafts. The acceptable criteria for 6-loci (HLA-A, -B antigen, -DRB1 allele) and 8-allele (HLA-A, -B, -C, -DRB1) donor-recipient HLA match, the evaluation of patients with donor-specific HLA antibodies, and the multiple determinants of unit quality are also reviewed in detail. Finally, a practical step-by-step guide to CB searches and the principles that guide ultimate graft selection are outlined.

Mid-Term Outcomes After Treatment for Antibody-Mediated Rejection by De Novo Donor-Specific HLA Antibody in Renal Transplant Recipients: Does Early Treatment Lead to Better Outcomes?

BackgroundDe novo donor-specific HLA antibody (DSA) and antibody-mediated rejection (ABMR) are strongly associated with late allograft loss in renal transplant recipients. However, the impact of therapeutic intervention with the current treatment options for ABMR remains unclear. This study aimed to elucidate the efficacy of treatment for ABMR. MethodsSixty-seven patients who had de novo DSAs underwent diagnostic biopsy for ABMR, and these patients were classified into 3 groups: ABMR-free group (n = 40), clinical ABMR group (n = 15), and subclinical ABMR group (n = 12). The ABMR-positive groups were treated mainly with double-filtration plasmapheresis followed by rituximab and corticosteroid pulse. The patient characteristics and graft outcomes were compared between groups. ResultsThe clinical and subclinical ABMR groups were younger and had a higher number and mean fluorescence intensity (MFI) of de novo DSAs than the ABMR-free group. The graft survival in the clinical ABMR group was significantly lower than that in the ABMR-free group, but the subclinical ABMR group had a surprisingly good graft survival rate compared to the ABMR-free group (43.3% vs 100% vs 94.2% 5 years after diagnostic biopsy in the clinical ABMR, subclinical ABMR, and ABMR-free groups, respectively, P < .001). ConclusionsOur findings indicated that early therapeutic intervention for patients with de novo DSAs may improve graft survival.

The suppressive effect on CD4 T cell alloresponse against endothelial HLA-DR via PD-L1 induced by anti-A/B ligation.

While donor-specific human leukocyte antigen (HLA) antibodies are a frequent cause for chronic antibody-mediated rejection in organ transplantation, this is not the case for antibodies targeting blood group antigens, as ABO-incompatible (ABO-I) organ transplantation has been associated with a favorable graft outcome. Here, we explored the role of CD4 T cell-mediated alloresponses against endothelial HLA-D-related (DR) in the presence of anti-HLA class I or anti-A/B antibodies. CD4 T cells, notably CD45RA-memory CD4 T cells, undergo extensive proliferation in response to endothelial HLA-DR. The CD4 T cell proliferative response was enhanced in the presence of anti-HLA class I, but attenuated in the presence of anti-A/B antibodies. Microarray analysis and molecular profiling demonstrated that the expression of CD274 programmed cell death ligand 1 (PD-L1) increased in response to anti-A/B ligation-mediated extracellular signal-regulated kinase (ERK) inactivation in endothelial cells that were detected even in the presence of interferon-γ stimulation. Anti-PD-1 antibody enhanced CD4 T cell proliferation, and blocked the suppressive effect of the anti-A/B antibodies. Educated CD25+ CD127- regulatory T cells (edu.Tregs ) were more effective at preventing CD4 T cell alloresponses to endothelial cells compared with naive Treg ; anti-A/B antibodies were not involved in the Treg -mediated events. Finally, amplified expression of transcript encoding PD-L1 was observed in biopsy samples from ABO-I renal transplants when compared with those from ABO-identical/compatible transplants. Taken together, our findings identified a possible factor that might prevent graft rejection and thus contribute to a favorable outcome in ABO-I renal transplantation.

Third-party vessel allografts in kidney and pancreas transplantation: Utilization, de novo DSAs, and outcomes.

Third-party vascular allografts (VAs) are an invaluable resource in kidney and pancreas transplantation when vascular reconstruction is needed and additional vessels from the organ donor are not available. We report the largest single-center experience to date on VA use, at a high-volume U.S. transplant center. Over a 7-year period, VAs were used for vascular reconstruction of 65 kidneys and 5 pancreases, in 69 recipients. The renal vein required reconstruction more often with right kidney transplantation (72.5% vs 27.5%, P<.001), and the renal artery required reconstruction more often with left kidney transplantation (67.6% vs 32.4%, P=.003). Eleven patients (15.9%) developed anti-VA de novo HLA donor-specific antibodies (dnDSAs) at a median time after transplantation of 19.0months. Higher number of HLA mismatches between the VA donor and the recipient, and development of anti-organ allograft dnDSAs were significant predictors of anti-VA dnDSA development. Those with anti-VA dnDSAs had a higher rate of organ allograft rejection (45.4% vs 13.8%, P=.03) compared to those without, but there was no significant difference in incidence of vascular complications or graft outcomes. VAs can help circumvent challenging surgical situations. Anti-VA dnDSAs do not adversely affect organ allograft outcomes; however, they can contribute to HLA sensitization in the recipients.

P1602MICROVASCULAR INFLAMMATION WITHOUT DSA OR C4D: AN ORPHAN CATEGORY IN BANFF CLASSIFICATION WITH INTENSE CAPILLARITIS AND CYTOTOXIC T-CELL INFILTRATION

Abstract Background and Aims Antibody-mediated rejection (ABMR) associated with donor-specific HLA antibodies (DSA) is the leading cause of late allograft failure after kidney transplantation. Microvascular inflammation (MVI) without detectable circulating DSA or C4d + cannot be classified as ABMR according to Banff-2017. The involvement of intragraft lymphocyte subsets in the development of humoral damage in kidney transplantation (KT) is relevant. We aimed to analyze lymphocyte subset distribution in kidney transplant biopsies (KTBx) with ABMR compared with MVI and with normal KTBx. Method KTBx with ABMR, MVI (g+ptc≥2, without DSA) or normal findings were included. DSA were identified with Luminex single antigen assays. Intragraft lymphocyte subsets’ characterization was performed by immunohistochemistry: T-lymphocytes (CD3, CD4, CD8, Foxp3), B-lymphocytes / plasmatic cells (CD20, CD138), NK cells (CD56), macrophages / monocytes (CD68), cytotoxic cells (TIA1) and activated cells (PD1) were evaluated. Results We analyzed 34 KTBx: 21 ABMR, 5 MVI and 8 KTBx with normal findings. KT with ABMR and MVI had more proteinuria at the time of the biopsy compared with the normal group (575 mg/24h and 964 mg/24h vs 147 mg/24h, p=0.002 and p=0.005 respectively). DSA were more frequently detected in patients with ABMR (95.2% vs 0% and 37.5%, p&amp;lt;0.001 and p=0.003 respectively). KTBx with ABMR and MVI had increased cytotoxic T-cell infiltration apparently corresponding to NK cells in peritubular capillaries (ptc) compared to normal group. Moreover, both groups showed a greater number of macrophages and monocytes in glomeruli. KT with MVI but not with ABMR had a significantly increased activated cell infiltration (PD1+) in ptc compared to the normal group, and showed an increased cytotoxic T-cell infiltration in glomeruli compared to ABMR and normal groups. Conclusion ABMR and MVI have an increased infiltration of NK cells with cytotoxic activity in ptc that differs from the normal group. However, KT with MVI show greater infiltration of activated cells in ptc and cytotoxic T-cell in glomeruli compared to ABMR suggesting the possibility of different activation pathways.

P1646SURVIVAL OF KIDNEY TRANSPLANTS ACCORDING TO PRESENCE OF HLA ANTIBODIES WHEN DONORSPECIFIC ANTIBODIES ARE SCREENED DURING POSTTRANSPLANT FOLLOW-UP

Abstract Background and Aims Posttransplant kidney survival depends on several risk factors. A careful immunogenetic matching and the absence of HLA donor specific antibodies (DSA) seem to determine the longevity of the transplant. Method Screening the presence of donor specific HLA antibodies in our posttransplant outpatients was implemented in 2010 (every 6 months in case of DSA free patients for two years, then yearly, and every 3 months in case of DSA + patients for two years, then twice a year). At the same time a treatment protocol was implemented, omitting reduction of immunosuppressive drugs in case of newly detected DSA, and most important with preventing steroid withdrawal in this case.The present single center study reports the long-term survival and kidney function from patients undergoing HLA-screening after transplantation between 2010 and 2016 with a follow-up until 2018. Using a Kaplan-Meier analysis patients without HLA antibodies (no HLA-ab), with HLA antibodies but without DSA (NDSA), and with donor-specific HLA antibodies (DSA) were compared by logrank-testing. Results A full dataset was obtained from 318 patients. The mean overall survival (patients and organ function) didn´t differ between the three groups, p=0.318: no HLA-ab 7.2 years (95%confidence interval 6.7;7.6), NDSA 6.6 (5.9;7.2), DSA 6.8 (6.1;7.5), overall 7.0 (6.6;7.3), events are given in Table1. Whereas the mean patient survival didn´t differ between the groups (p=0.715), the mean death-censored graft survival differed significantly, p=0.008, with a reduced transplant survival in the patients with HLA antibodies but without donorspecific antibodies: no HLA-ab 8.0 years (95%confidence interval 7.7;8.3), NDSA 7.0 (6.4;7.6), DSA 7.6 (7.1;8.2), overall 7.7 (7.4;8.0), numbers are given in Table1. Conclusion In conclusion, the presence of HLA antibodies was associated with a reduced transplant survival. Patients with HLA antibodies had a worse survival than patients with DSA undergoing HLA screening with a personalised immunosuppressive regimen. Immunosuppressive regimen of the groups, as well as other known risk factors of graft survival have to be further analysed. The results of these multivariate analyses have to be awaited to determine whether the risk for graft loss inferred by HLA antibodies is independent from other factors.

P1776HLA TYPING BY NGS ENHANCES MISMATCH DETECTION IN LIVING KIDNEY TRANSPLANTATION

Abstract Background and Aims Antibody mediated rejection (ABMR) due to the development of donor specific HLA antibodies is the most common cause for graft loss in kidney transplant recipients. Therefore, a low count of HLA mismatches is favorable in the course of transplantation. Hitherto, several techniques for HLA typing have been adapted in parallel, e.g. serological or low-resolution PCR. In this single-center study, we aimed to perform “HLA re-typing” by next generation sequencing (NGS) in a living kidney transplant cohort (n=143), in order to identify HLA mismatches in donor/recipient pairs, that were missed by previous HLA typing methods. Method We isolated DNA of 102 donor/recipient pairs, that received a living kidney transplantation at the University Hospital Leipzig (Germany) between 1998 and 2018, and performed long range PCR of all known HLA loci. Using these NGS results, we compiled a genomic HLA mismatch formula. We evaluated, if genomic HLA typing identifies more HLA mismatches compared to the results of previous HLA typing methods, that were recorded in the Eurotransplant database for our donor/recipient pairs. Results By genomic HLA typing, we were able to identify HLA mismatches in 10.2 % of donor/recipient pairs, that have been missed by previous methods. In our cohort, we recognized most mismatches in the DRB1 locus, that were missed with other HLA typing techniques. Conclusion Our results suggest, that genomic HLA typing can be more precise in detecting HLA mismatches in donor recipient/pairs. Especially in the course of living kidney transplantation, a higher accuracy in HLA typing might enhance donor selection. Furthermore, accurate HLA typing can influence graft management in the course of ABMR, as it may improve the detection of donor specific HLA directed antibodies.

Non-donor specific anti-human leukocyte antigen (HLA) antibodies are not associated with poor outcome in hematopoietic stem cell transplant recipients

Testing for anti-human leukocyte antigen (HLA) antibodies has now become standard practice in allogeneic hematopoietic stem cell transplantation (HSCT), and anti-HLA antibodies (both donor specific and non-donor specific) are being identified and have many potential consequences. Most studies suggest that donor-specific HLA antibodies lead to adverse outcomes, though little is reported on non-donor specific anti-HLA antibodies. We present the results of a retrospective cohort analysis of 157 patients who received HSCT at the University of Rochester over a period of four years. We identified 45 patients (28.7%) who had detectable anti-HLA antibodies, while only one patient (0.6%) had donor-specific anti-HLA antibodies. Patients with prior pregnancies and multiple transfusions were at increased risk to develop antibodies. In our cohort, the presence of non-donor specific anti-HLA antibodies did not significantly impact overall survival, progression free survival, graft failure, or transplant-related mortality.

Antibody-induced vascular inflammation skews infiltrating macrophages to a novel remodeling phenotype in a model of transplant rejection.

HLA donor-specific antibodies (DSAs) binding to vascular endothelial cells of the allograft trigger inflammation, vessel injury, and antibody-mediated rejection (AMR). Accumulation of intragraft-recipient macrophages is a histological characteristic of AMR, which portends worse outcome. HLA class I (HLA I) DSAs enhance monocyte recruitment by activating endothelial cells and engaging FcγRs, but the DSA-activated donor endothelial influence on macrophage differentiation is unknown. In this study, we explored the consequence of DSA-activated endothelium on infiltrating monocyte differentiation. Here we show that cardiac allografts from murine recipients treated with MHC I DSA upregulated genes related to monocyte transmigration and Fc receptor stimulation. Human monocytes co-cultured with HLA I IgG-stimulated primary human endothelium promoted monocyte differentiation into CD68+ CD206+ CD163+ macrophages (M(HLA I IgG)), whereas HLA I F(ab')2 stimulated endothelium solely induced higher CD206 (M(HLA I F(ab')2 )). Both macrophage subtypes exhibited significant changes in discrete cytokines/chemokines and unique gene expression profiles. Cross-comparison of gene transcripts between murine DSA-treated cardiac allografts and human co-cultured macrophages identified overlapping genes. These findings uncover the role of HLA I DSA-activated endothelium in monocyte differentiation, and point to a novel, remodeling phenotype of infiltrating macrophages that may contribute to vascular injury.

Low Immunologic Risk Living Related Renal Transplant Using Very Low-Dose Antithymocyte Globulin as Induction Therapy: A Single Tertiary Hospital Experience

The aim of induction therapy in the management of kidney transplant is to reduce the incidence of acute rejection and delayed graft function after kidney transplant. The agent for induction therapy differs depending on the recipient risks. The regimen can be either polyclonal (rabbit antithymocyte globulin [rATG]) or monoclonal antibody (basiliximab). Basiliximab is commonly used in patients with low immunologic risk. However, to date we know that the use of rATG on T cell depletion is dose dependent and more potent antirejection therapy. Therefore, we would like to look at 1-year graft function of very low-dose rATG in low immunologic risk recipients.All low immunologic risk patients who received low-dose rATG (0.5 mg/kg of body weight daily) during transplant (day 0) and on days 1 and 2 were recruited. Their renal function, HLA donor-specific antibodies, lymphocyte counts, protocol biopsy results, and cytomegalovirus (CMV) polymerase chain reaction were monitored as per clinical practice.All 10 patients had immediate graft function. Low-dose rATG caused lymphocyte counts to deplete immediately on day 0, and the effect lasted about 1 month post-transplant. All the patients had stable graft function without any significance episode of rejection. Only one patient had de novo HLA-DQ antibody. It is good to know that without prophylaxis antiviral in CMV+ donor to CMV+ recipient, the incidence of CMV viremia is considerably low in our cohort.Very low-dose rATG is an effective induction immunosuppression in low immunologic risk patients with acceptable infection risk.

One-Year Outcomes of Living Related Kidney Transplant in Patients With Preformed HLA Donor-Specific Antibodies: A Single-Center Experience in Malaysia

The shortage of deceased donors led to an increase of living related renal transplant performed in the presence of donor-specific antibodies (DSAs) or ABO incompatibilities. There are various desensitization protocols that have been proposed. Here, we describe the outcome of these sensitized patients.This is a prospective cohort study recruiting all kidney transplant recipients from August 2016 until June 2018. Deceased donations, ABO incompatible patients, and sensitized patients who were not prescribed on our desensitization protocol were excluded. Recipients were screened for the presence of HLA-antibodies 1 month before transplant. Those with positive DSA will undergo flow cytometry (risk stratification). We are using a protocol that consisted of intravenous rituximab 200 mg (day -14), intravenous antithymocyte globulin 5mg/kg (day 0-4), plasma exchange post transplant for patients with mean fluorescent intensity (MFI) < 3000, and negative flow cytometry. Those patients with MFI ≥ 3000 or positive flow cytometry need extra cycles pretransplant.A total of 40 patients were recruited, and 20 were sensitized patients. Among the sensitized group 4 of 20 had flow cytometry crossmatch positive, while all had preformed HLA-DSA. A total of 8 of 20 had class I HLA-DSA, 11 of 20 had class II HLA-DSA, and 1of 20 was positive for both class I and II HLA-DSA. Mean immunodominant MFI was 2133.4 (standard deviation [SD], 4451.24) and 1383.7 (SD, 2979.02) for class I and class II, respectively. At 1 year, mean serum creatinine was 108.90 (SD, 25.95) and 118.42 (SD, 31.68) in sensitized and unsensitized patients, respectively. One of 20 unsensitized patients had Banff 1B rejection at 3 months, and there was no significant rejection in sensitized patients at 6 months and 1 year. There was no difference in the occurrence of de novo HLA-DSA between the groups.Desensitization protocols may help to overcome incompatibility barriers in living donor renal transplant. The combination of low-dose rituximab, antithymocyte globulin, and judicious use of plasma exchange has worked well for our cohort.

Donor-specific B Cell Memory in Alloimmunized Kidney Transplant Recipients: First Clinical Application of a Novel Method.

HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell-derived HLA antibodies (DSA-M) in renal allograft recipients with pretransplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (AMR) using a recently developed method. Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells were collected at 3 timepoints (pretransplant, mo 6, mo 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated peripheral blood mononuclear cells using SAB assays and compared HLA antibody profiles with same day plasma results. Plasma SAB analysis revealed 35 DSA in 20 patients pretransplant. DSA-M were detected in 9 of 20 (45%) patients and for 10 of 35 specificities (29%). While median mean fluorescence intensity values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3 of 6 patients with AMR and low mean fluorescence intensity DSA (<3000) had DSA-M. Overall, pretransplant DSA/DSA-Mpos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical AMR (P = 0.032) and a higher extent (g≥1 + ptc≥1) of microvascular inflammation (67% vs 9%, P = 0.02). In 17 patients (28 DSA) with posttransplant analyses, persisting DSA posttransplant had more often DSA-M (6/12; 50%) than nonpersisting DSA (2/16; 13%). Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pretransplant risk stratification in patients with DSA.

Short-term Immunopathological Changes Associated with Pulse Steroids/IVIG/Rituximab Therapy in Late Kidney Allograft Antibody Mediated Rejection.

B-cell depletion is a common treatment of antibody-mediated rejection (ABMR). We sought to determine the specific immunopathologic effects of this therapeutic approach in kidney transplantation. This was a prospective observational study of kidney transplant recipients diagnosed with late ABMR (>3 months after transplant). Patients received treatment with pulse steroids, IVIG, and rituximab. Donor specific HLA antibodies (DSA), kidney allograft pathology, renal function, immune cell phenotypes, and 47 circulating cytokines were assessed at baseline and at three months. We enrolled 23 patients in this study between April 2015 and March 2019. The majority of patients were male (74%) and Caucasian (78%) with an average age of 45.6±13.8 years. ABMR was diagnosed at 6.8±5.9 years (4 months-25 years) post-transplant. Treatment was associated with a significant decline in circulating HLA class I DSA (P=0.003) and class II DSA (P=0.002) and peritubular capillaritis (ptc, P=0.04) compared to baseline. Serum creatinine, BUN, eGFR, and proteinuria (UPC) remained stable. Circulating B-cells were depleted to barely detectable levels (P≤0.001), whereas BAFF (P=0.001), APRIL (P<0.001), and IL-10 (P=0.02), levels increased significantly post-treatment. Notably, there was a significant rise in circulating CD4+ (P=0.02) and CD8+ T-cells (P=0.003). We also noted a significant correlation between circulating cytotoxic CD8+ T-cells and BAFF (P=0.05), regulatory T-cells and IL10 (P=0.002), and HLA class I DSA (P=0.005). Short-term pulse steroids/IVIG/rituximab therapy was associated with inhibition of ABMR (DSA and ptc), stabilization of kidney function, and increased regulatory B-cell and T-cell survival cytokines. Additional studies are needed to understand the implications of B cell-depletion on the crosstalk between T-cells, B-cells, and humoral components that regulate ABMR.