Donor HLA Typing Research Articles

Resolving Ambiguities in Deceased Donor HLA Typing: Next Generation Sequencing’s Role in DPB1 Allele Discrimination and Immunological Implications

Abstract HLA typing is crucial for assessing immunological compatibility between donors and recipients before transplantation. Deceased donor (DD) typing, typically via real-time PCR (rtPCR), provides rapid results vital for organ allocation. rtPCR primarily targets exons encoding the highly polymorphic peptide binding region (PBR) of HLA molecules. rtPCR cannot target all polymorphic sequences. Consequently, several possibilities of allele combinations are provided, leading to ambiguous typing results. As recent evidence suggests antibody responses can target regions in exons outside the PBR, notably in Exon 3 of the DPB1 locus, typing ambiguities can lead to the inability to assess histocompatibility between a donor and recipient. Herein, the aim was to investigate the utility of Next Generation Sequencing (NGS) in resolving DPB1 ambiguities in DD typing. A retrospective analysis of DD typing from February 2022 to January 2024 examined instances where NGS was used to resolve ambiguities reported via rtPCR. Each case was reviewed to understand the reason for resorting to NGS typing. The study focused on DPB1 ambiguities and assessed whether NGS effectively reduced ambiguity and differentiated alleles with polymorphisms outside the PBR. In total, 29 DD cases were found to have NGS testing results. In 14 cases, NGS was specifically performed to resolve DPB1 ambiguities. The ambiguities were significantly reduced from hundreds detected in rtPCR to 4 or fewer possible allele combinations with NGS. Eleven cases demonstrated polymorphism in exons outside the PBRs. Of these, only 2 cases showed no typing ambiguity with NGS. In the remaining 9 cases, though polymorphisms could be resolved in the PBR, ambiguities remained in exon 3, specifically at codon 96. In these cases, NGS could not distinguish lysine (K) from arginine (R). For example, DPB1*835:01 with the 96R motif could not be distinguished from DPB1*34:01with the 96K motif. The current investigation found that NGS cannot resolve all ambiguities at position 96, likely due, at least in part, to persistent cis-trans ambiguities. Our results suggest that although NGS drastically limited the number of ambiguities in 9 of 11 cases, it still could not adequately distinguish all immunologically relevant alleles. Inability to resolve the 96R/K epitope, which has been shown to be immunogenic, could impact solid organ transplantation, potentially leading to rejection in mismatched recipient-donor pairs at this position. Sequencing methods that generate longer reads, such as nanopore, may help resolve some of these cases if they are indeed due to cis-trans ambiguities.

HLA-B*51:11N revealed by NGS in confirmatory deceased donor HLA typing

HLA-B*51:11N revealed by NGS in confirmatory deceased donor HLA typing

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.

HLA-DRB1 Hed Is Associated with Improved Survival and Decreased Relapse in Patients with Hematologic Malignancies Following Allogeneic Hematopoietic Stem Cell Transplant

Introduction: Despite advances in disease monitoring and maintenance therapies, the risk of relapse following allogeneic hematopoietic cell transplant (HCT) remains a major concern. Attention has turned to understanding how diversity across the immune system can influence tumor surveillance. Due to their protein's integral role in binding and presenting antigens to immune cells, HLA genes have been a major focus because a high number of polymorphisms alter their peptide-binding grooves and impact their repertoire of antigens. Based on the heterozygote advantage hypothesis, heterozygous HLA genotypes were thought to better elicit immune responses due to a higher probability of binding antigens. Further refinement of HLA diversity centers on the immunopeptidome and variations in peptide-binding specificities. HLA evolutionary divergence (HED) represents a quantitative measure of physiochemical sequence divergence between peptide-binding grooves encoded by HLA alleles. Greater sequence divergence across HLA alleles leads to a more expansive repertoire of antigens and helps to explain that high HED was associated with improved overall survival (OS) in patients with solid tumors treated with immune checkpoint inhibitors (Chowell et al. Nat Med, 2019). With a more diversified immune response, we hypothesize that high HED will improve tumor surveillance and impact survival and relapse following allogeneic HCT. Methods: Patients who underwent 8/8 HLA-A, -B, -C and -DRB1 matched first allogeneic HCT for AML, MDS, ALL, CML, or lymphoma between 2008-2018 were identified from the CIBMTR database. All patients received myeloablative conditioning, except for those patients with lymphoma. Using donor HLA typing to the allele level and reference amino acid sequences for peptide binding domains, HLA HED was calculated for each HLA class I allele (-A,-B,-C) and HLA class II allele (-DRB1) using the Grantham Distance metric (Pierini, et al. Mol Biol Evol, 2018). An overall mean HED score was calculated for HLA class I and HLA-DRB1 alleles and was initially divided at the median to classify high versus low HED. HED was then evaluated based on quartiles with high HED defined as overall mean HED greater or equal to the upper quartile. The primary outcomes were OS and relapse. Secondary outcomes included transplant-related mortality (TRM), disease-free survival (DFS), and graft-versus-host disease (GVHD). Multivariate analysis using Cox proportional hazards model was performed with factors violating the proportional hazards assumptions adjusted through stratification. P-value ≤ 0.01 was significant for this study. Results: A total of 9,231 patients received an 8/8 HLA-matched allogeneic HCT from 2008-2018 according to selection criteria. With high HED defined by the median, there was no association with OS and HLA class I HED (HR 1.00; 95% CI 0.93 - 1.07, P=0.935) or with HLA-DRB1 HED (HR 0.96; 95% CI 0.91 -1.02, P=0.225). Relapse was not associated with HLA class I HED (HR 1.04, 95% CI 0.96-1.12, P=0.354) or with HLA-DRB1 HED (HR 0.99, 95% CI 0.93 - 1.06, P=0.857). There were no significant associations with HED and the secondary outcomes of TRM, DFS, or GVHD. HED was then characterized by quartiles. High HLA-DRB1 HED (upper quartile) was associated with improved OS (HR 0.91; 95% CI 0.85 - 0.96, P=0.0019) and decreased relapse (HR 0.87; 95% CI 0.80 - 0.95, P=0.0014) when compared to low HED (Q1-Q3). Improved DFS was observed with high HLA-DRB1 HED but failed to achieve statistical significance (HR 0.92; 95% CI 0.86 - 0.98, P=0.011). There was no association between HLA class I HED and OS, relapse, or DFS. Conclusions: High HLA-DRB1 HED, defined as upper quartile, was associated with improved OS and decreased relapse in patients with hematologic malignancies following allogeneic HCT. High HED promotes a diverse repertoire of antigens, which may heighten tumor surveillance and enhance graft versus leukemic effect. Since similar associations were not replicated when high HED was defined by the median, the effects of HLA-DRB1 HED are most likely observed in the most divergent donor-recipient HLA pairings. Previous work has implicated the role of HLA class II alleles in affecting immune function and contributing to relapse following HCT. Therefore, HLA-DRB1 may play a more integral role in tumor surveillance post-HCT, which can have important implications in donor selection, especially in HLA mismatched settings.

Evaluation of GenDx Protocol for HLA NGS Genotyping Using the Ion Torrent Sequencing Platform

Abstract Background: The Human Major Histocompatibility Complex (HLA) is the most polymorphic region of the human genome and encodes molecules with a central role in antigen-specific immune responses. Class I and class II HLA genes have an important role in bone marrow and solid organ transplantations. HLA typing by Next Generation Sequencing (NGS) provides unambiguous second field or allelic level resolution and, therefore, better matching of donor and recipient and a higher chance of survival for transplanted patients. The aim of this study was to validate GenDx NGS HLA typing protocol for 6 HLA loci (HLA-A, -B, -C, DRB1, -DQB1 and DPB1). Materials and methods: 240 samples of both bone marrow healthy donors from the Bulgarian Bone Marrow Donors Registry (BBMDR) and bone marrow recipients were sequenced on the Ion Torrent System using the GenDx NGS HLA typing kit, compared to 65 samples from the BBMDR analyzed by Holotype HLA kit (Omixon) and sequenced on the Illumina platform MiniSeq. A number of metrics including allele balance, read length, mappability, coverage and ambiguity were assessed in order to evaluate the GenDx NGS HLA typing protocol. We also investigated haplotype and allele frequencies of class I (HLA-A, -B, -C) and class II (HLA-DRB1, -DQB1 and -DPB1) alleles. Results: The results demonstrate the accuracy of this protocol, eliminating almost all ambiguities, providing a reasonable read length of 180, 76% mappability and 1267 depth of coverage on average for all 6 HLA loci. We found that the most frequent alleles for class I are HLA-A*02:01:01 (27,5%), B*51:01:01 (16,5%), C*07:01:01 (16,5%) and class II – DRB1*11:04:01 (14,5%), DQB1*03:01:01 (26,9%) and DPB1*04:01:01 (33,2%) which corresponds with other investigations of the research team on HLA diversity in the Bulgarian population. Conclusion: The main advantages of GenDx NGS HLA typing protocol are the shorter time for DNA library preparation and the shorter sequencing time. Phasing ambiguities are also liminated, which is a significant advantage in the clinical environment. The presented data show the suitability of this NGS protocol for clinical practice, especially in HLA typing of donors from Bone Marrow Donors registries.

O21 Estimating rates of immunologically incompatible organ offers caused by use of antigen-level deceased donor HLA typing for allocation

O21 Estimating rates of immunologically incompatible organ offers caused by use of antigen-level deceased donor HLA typing for allocation

O32 Bioinformatic segregation of chimeric libraries enables accurate donor HLA typing from allograft biopsies by ONT

O32 Bioinformatic segregation of chimeric libraries enables accurate donor HLA typing from allograft biopsies by ONT

P220 Managing STAT deceased donor HLA typing and laboratory staff shortage: Experience of two HLA laboratories

P220 Managing STAT deceased donor HLA typing and laboratory staff shortage: Experience of two HLA laboratories

Chronic myeloid leukemia following low dose radioactive iodine (I131) for treatment of papillary thyroid cancer

Introduction: Therapy-related myeloid neoplasms result from exposure to specific agents such as ionizing radiation, chemotherapy, and immunosuppressive agents. Radioactive iodine plays an essential role in managing differentiated thyroid cancers such as papillary thyroid carcinoma to reduce the risk of recurrence. Patient concerns: We present the case of a 48-year-old Bangladeshi male with a history of papillary thyroid cancer after total thyroidectomy and radiofrequency ablation therapy with I131 (32.6 mCi) who presented with abdominal pain and was found to have chronic myeloid leukemia 3 years after treatment for papillary thyroid cancer. Diagnosis: Chronic myeloid leukemia secondary to low-dose radioactive iodine (I131). Interventions: The patient was started on dasatinib (140 mg/day). Outcomes: The patient is currently being prepared for bone marrow transplantation by donor search and HLA typing. Conclusion: This case emphasizes the importance of considering the leukemogenic effects of I131.

Clinical significance of low pre-transplant donor specific antibodies (DSA) in living donor kidney recipients with negative complement-dependent cytotoxicity crossmatches (CDCXM), and negative flow cytometry crossmatches (FLXM) – A single-center experience

BackgroundIt is controversial whether all donor-specific antibodies (DSA) detected by the solid-phase single antigen bead (SAB) assay negatively affect kidney transplantation outcomes. The study aimed to evaluate the possible clinical significance of low pre-transplant DSA in living donor kidney recipients. We analyzed a group of patients with HLA-A, B, and -DR DSA reactivities below a virtual crossmatch (VXM) value of 5000 MFI but with all VXM DSA reactivities at HLA-DQ, -DP, and -Cw, which were not typed routinely for donors prior to transplantation. We also investigated the incidence of persistent and de novo DSAs in available posttransplant SAB assays. MethodsFrom the historical cohort of living donor recipients transplanted between 2014 and 2018 at our center (n = 82), 55 patients met the inclusion criteria, namely: these patients were > 18 years old with non-HLA identical sibling donors, who were not desensitized, who had available pre-transplant SAB results, and who had negative both complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FLXM) results. An additional donor HLA typing, performed for all 55 recipients, identified donor additional HLA-DQ, -DP, and -Cw DSA reactivities. These patients were then divided by SAB reactivity into three groups: 1) those with DSA-positive reactivities; 2) those with non-donor-specific anti-HLA reactivities (NDSA); and, 3) those who were anti-HLA-negative. All these recipients were followed for three years and checked for their de novo or persistent DSA. ResultsIn the studied cohort, DSA-positive, NDSA reactive, and anti-HLA negative recipients constituted 33%, 36%, and 31% of 55 patients, respectively. Non-routinely considered pre-transplant HLA-DQ, -DP, and -Cw DSA-positive reactivities were shown in as many as 78% of DSA-positive cases (group 1) with the lowest MFI value of 319 to DP4 and the highest MFI of 5767 to DQ2. Of the pre-transplant HLA-A, B, and -DR DSA reactivities, only -DR52 DSA reactivity reached the highest MFI value of 2191. These detected DSAs did not reduce the mean estimated glomerular filtration rate (eGFR) values and did not increase the incidence of proteinuria in recipients. While the 3-year graft survival was lower in the DSA-positive group (94.4%) with one recipient who lost kidney transplant, the difference was not significantly different (p = 0.7) from the NDSA (100%) and negative (100%) groups. In terms of the incidence of de novo acute antibody-mediated rejection (AMR) at three years after transplantation, no case has been reported in the cohort. This may suggest that low DSA-positive recipients do not experience higher rejection rate. However, DSA-positive recipients had a tendency for a higher frequency of C4d deposits in peritubular capillaries (PTC) and de novo DSA. ConclusionOur 3-year follow-up of patients with low pre-transplant DSA found no association with a deterioration in graft function and worse graft survival. Furthermore, we did not observe an increase in AMR in our patients with low DSA. A larger cohort and a longer follow-up period may be needed to evaluate the tendency of low DSA-positive recipients towards the higher incidence of C4d deposits in PTC and/or de novo DSA.

Single-cell TCR and mRNA sequencing of antigen-specific T cells reveal spatial trajectories from single time points with HLA and VDJ bias

Abstract Yellow fever (YF) vaccination induces massive CD8 expansion, comprising up to 10% of all circulating CD8 T cells. We hypothesize that HLA type and VDJ gene usage implicate the highly biased response towards certain immunodominant epitopes. PBMCs were collected from healthy human donors 3 weeks after vaccination with the yellow fever vaccine, Stamaril, and enriched for YF-CD8 T cells using HLA class I tetramers. We focused on the immunodominant epitopes NS4B and NS2A, and the less dominant epitope NS3. Cells were analyzed by single-cell T cell receptor (TCR) and mRNA sequencing, yielding detailed gene phenotypes for around 2000–3000 quality cells from each donor. Data was analyzed using SeqGeq, VDJ explorer, Seurat and Monocle2. From three donors, we performed in depth analyses on both TCR gene use bias across epitope specificity, and established pseudotime cell trajectories from a single sampling time point. We found that within the CD8 population from the same donor, VDJ usage was highly biased for the target antigen. Based on mRNA expression of a panel of 259 T-cell relevant genes, we performed unsupervised clustering, that shows cells with different epitope targets cluster interchangeably, indicating similar functional patterns. However, cells specific for the immunodominant epitopes had lower TCR clonal divergence between naïve-like and activated clusters, indicating a higher degree of clonal expansion compared to less immunodominant clones. Taken together, we show antigen-driven T cell responses are biased based on donor HLA-types, which could implicate development and success of T-cell driven immunotherapies. Supported by grants from Independent Research Fund Denmark (1029-00004B)

Prospective multi-centric study to analyze pre-transplant compatibility algorithm for live-related donor kidney transplant in Indian setting: the “Delhi approach”!

BackgroundSince no single test is always accurate and sensitive, two or more tests are used to increase the precision of evaluation. Different algorithms have been proposed by centers in Leiden, Basel, Vienna and Minnesota, etc. With an intention to develop an optimal algorithm for India, we evaluated pre-transplant compatibility tests for live-donor kidney transplants. Three tests complement dependent cyto-toxicity cross-match (CDCXM), flow-cytometry cross-match (FCXM) and anti-HLA antibody screening (HAS) were performed and confirmed by the anti-HLA antibody identification (HAI) assay in a multi-centric trial (three transplant centers) in India. Materials and methodsAll prospective recipients (and their potential donors) underwent low-resolution HLA typing as well as CDCXM, FCXM and HAS assays. In addition, HAI {single antigen bead assay; (SAB)} was done for all recipients to identify possible anti-HLA antibodies. In a virtual cross-match (VXM), antibody specificity was mapped to donor HLA type to determine donor-specific antibodies (DSA). Only patients without DSA were cleared for the transplant. Alternatively, patients with DSA were offered an exchange in the kidney paired donation (KPD) program. The screening results (CDCXM, FCXM, and HAS) were analyzed, individually as well as in combination of screening assays (CDCXM+HAS, CDCXM+FCXM, and FCXM+HAS) and the results were compared with those from the HAI test. ResultsOut of 100 patients, 69 were males and 31 were females; 85 recipients (85%) underwent a kidney transplant. The sensitivity of CDCXM was only 12.1% and the specificity of CDCXM was 100%; whereas the sensitivity of FCXM was 84.8% and the specificity of FCXM was 89.6%. The sensitivity and specificity of class I HAS was 88.2% and 84.3%, respectively. The sensitivity and specificity class II HAS was 88.0% and 80.0%, respectively. However, when both class I/II HAS were tested together the sensitivity increased to 97.0% and the specificity to 82.1%. Similarly, the sensitivity of combined FCXM+HAS had the sensitivity of 100% and the specificity of 76.1%; CDCXM+FCXM had the sensitivity of 84.8% and the specificity of 89.6% and CDCXM+HAS assays reached 97% with the specificity of 82.1%. ConclusionsOur results showed that the algorithm of FCXM with HAS produced the best sensitivity of 100%. The specificity of 76.1% indicate that the combined FCXM+HAS assays may detect up to 24.9% false positive results. We suggest that these false-positives may be easily resolved by performing the virtual crossmatch based on HAI (SAB) results. In our reflex testing algorithmic approach only 49% patients needed HAI (SAB). Finally, our results suggested that the CDCXM assay may be discontinued in pre-transplant workup owing to its very low sensitivity (12.1%).

Donor HLA genotyping of ex vivo expanded urine cells from kidney transplant recipients.

Antibody-mediated rejection (AMR) induced by donor-specific anti-HLA antibodies (DSA) remains a major cause of long-term graft loss after kidney transplantation. Currently, the presence of DSA cannot always be determined at a specific allele level, because existing donor HLA typing is low resolution and often incomplete, lacking HLA-DP, and occasionally HLA-C and HLA-DQ information and historical donor DNA samples are not available for HLA retyping. Here we present a novel, non-invasive technique for obtaining donor DNA from selectively expanded donor cells from urine of renal transplant recipients. Urine-derived cells were successfully expanded ex vivo from 31 of 32 enrolled renal transplant recipients, and with DNA obtained from these cells, donor HLA typing was unambiguously determined for HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 loci by next-generation sequencing. Our results showed 100% concordance of HLA typing data between donor peripheral blood and recipient urine-derived cells. In comparison, HLA typing showed that DNA derived from urine sediments mainly contained recipient-derived DNA. We also present the successful application of our novel technique in a clinical case of AMR in a renal transplant recipient. Urine-derived donor cells can be isolated from kidney transplant recipients and serve as a suitable source of donor material for reliable high-resolution HLA genotyping. Thus, this approach can aid the assessment of DSA specificity to support the diagnosis of AMR as well as the evaluation of treatment efficacy in kidney transplant recipients when complete donor HLA information and donor DNA are unavailable.

BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre‐transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation.

The correlation between post-allo-HSCT CMV infection and the difference affinity of donor HLA-type recognition of CMV antigen peptide in children

目的探索儿童异基因造血干细胞移植（allo-HSCT）后供者HLA型别识别不同巨细胞病毒（CMV）抗原肽亲和力与发生CMV感染的相关性。方法回顾性分析武汉儿童医院血液肿瘤科146例allo-HSCT患儿移植后6个月内发生CMV再激活、CMV感染、CMV组织器官病的数据，分析其与供者HLA型别识别CMV抗原肽差异的相关性。结果纳入研究的146例移植后患儿中HLA型别与PP65单独高亲和力82例（56.16％），移植后发生CMV感染34例（41.46％）；IE-1高亲和力型5例，移植后感染0例；无明确亲和力多肽2例，感染0例；PP65+IE-1高亲和力型52例，移植后感染13例（25.00％）；与PP65和PP50共同高亲和力型5例，移植后感染3例；未遇到仅与PP50高亲和力HLA供者。与仅携带PP65高亲和力等位基因组比较，伴有IE-1高亲和力HLA位点供者移植后表现出更低的CMV感染发生率（22.81％对41.46％，P＝0.029）。结论CMV PP65和IE-1高亲和力型别HLA类型覆盖约99.8％的供者，选择CMV抗原肽IE-1高亲和力型别HLA供者或可降低儿童血液病患者allo-HSCT后CMV感染风险。

Missing Self-Induced Microvascular Rejection of Kidney Allografts: A Population-Based Study.

Circulating anti-HLA donor-specific antibodies (HLA-DSA) are often absent in kidney transplant recipients with microvascular inflammation (MVI). Missing self, the inability of donor endothelial cells to provide HLA I-mediated signals to inhibitory killer cell Ig-like receptors (KIRs) on recipient natural killer cells, can cause endothelial damage in vitro, and has been associated with HLA-DSA-negative MVI. However, missing self's clinical importance as a nonhumoral trigger of allograft rejection remains unclear. In a population-based study of 924 consecutive kidney transplantations between March 2004 and February 2013, we performed high-resolution donor and recipient HLA typing and recipient KIR genotyping. Missing self was defined as the absence of A3/A11, Bw4, C1, or C2 donor genotype, with the presence of the corresponding educated recipient inhibitory KIR gene. We identified missing self in 399 of 924 transplantations. Co-occurrence of missing self types had an additive effect in increasing MVI risk, with a threshold at two concurrent types (hazard ratio [HR], 1.78; 95% confidence interval [95% CI], 1.26 to 2.53), independent of HLA-DSA (HR, 5.65; 95% CI, 4.01 to 7.96). Missing self and lesions of cellular rejection were not associated. No HLA-DSAs were detectable in 146 of 222 recipients with MVI; 28 of the 146 had at least two missing self types. Missing self associated with transplant glomerulopathy after MVI (HR, 2.51; 95% CI, 1.12 to 5.62), although allograft survival was better than with HLA-DSA-associated MVI. Missing self specifically and cumulatively increases MVI risk after kidney transplantation, independent of HLA-DSA. Systematic evaluation of missing self improves understanding of HLA-DSA-negative MVI and might be relevant for improved diagnostic classification and patient risk stratification.

KIR- Ligand Mismatching Is Associated with Inferior Survival in Patients with Acute Leukemia Following T-Cell Replete Peripheral-Blood Stem Cell Haplo-Identical Transplantation with Post-Transplant Cyclophosphamide. a Study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT)

Background: Haplo-identical stem cell transplantation is increasingly been used in recent years in the treatment of acute leukemia. In particular, the use of T-cell replete grafts with post-transplant cyclophosphamide (PTCy) for prevention of graft-vs-host disease (GVHD) is becoming a valid treatment option with encouraging outcome. The initial studies used bone-marrow as the stem-cell source, but more recently, peripheral blood stem cells (PBSC) are also used. There is more limited data on the prognostic factors following haplo-identical transplantation with PBSC. Natural killer (NK) allo-reactivity as predicted by missing killer-cell immunoglobulin-like receptor (KIR) ligands in the recipients that are present in the donors has been shown to be an important factor in the outcome of T-cell depleted haplo-identical transplants. NK alloreactivity is associated with reduced relapse risk and improved survival in patients with AML, but not in patients with adult ALL. In addition, patients with donor NK alloreactivity had better engraftment and lower risk of GVHD. The role of NK cell allo-reactivity in the setting of T-cell replete haplo-identical transplant with PBSC and PTCy is unknown.Patients and Methods: The study included 238 patients, 139 male, 99 female, median age 45 years (range, 18-78) with AML (n=176) or ALL (n=62) who had T-replete haplo-identical transplant with PTCy between the years 2010-2015. All patients were given PBSC as their stem cell source. Patients were in CR1 (38%), CR2 (24%) or active disease (38%). Conditioning was myeloablative (47%) or reduced-intensity (53%). GVHD prevention included cyclosporine or tacrolimus with mycophenolate, in addition to PTCy, in 82%. KIR-ligand mismatching was predicted according to patient and donor HLA typing and correlated with transplantation outcomes. In all, 89 patients (37%) had a KIR mismatch in the GVHD direction, based on missing expression of C1, C2 or Bw4 ligands in the recipients that were expressed in their donors. There was no significant difference in patient characteristics between the group with and without KIR mismatch.Results: The median follow-up was 14 months (range, 1-50). Ninety-four percent of patients engrafted. The 2-year relapse and non-relapse mortality (NRM) rates in the entire group were 36% (95%CI, 29-42) and 22% (17-27), respectively. The leukemia-free (LFS) and overall survival (OS) rates were 43% (36-50) and 49% (42-56), respectively. Acute GVHD grade II-IV and chronic GVHD occurred in 34% (29-40) and 32% (26-39), respectively. The 2-year OS was 35% (22-47) in patients with KIR mismatch and 51% (42-60) in patients with no KIR-ligand mismatching (P=0.02). 2-year LFS rates were 27% (16-38) and 47% (38-56), respectively (P=0.003). Multivariate analysis identified AML (compared to ALL) (HR 0.51, P=0.008), active disease at transplantation (HR 5.03, P<0.001) and KIR mismatching (HR 1.60, P=0.03) as factors associated with 2-year OS. A center effect was also identified (P=0.05). The same factors predicted for LFS. KIR mismatching was associated with a higher incidence of relapse, 47% (34-58) and 32% (24-41), respectively (P=0.05). Multivariate analysis identified active disease at transplantation (HR 6.38, P<0.001), KIR mismatching (HR 1.97, P=0.03), female donors (HR 0.58, P=0.03) and reduced-intensity conditioning (HR 1.71, P=0.05) as factors associated with 2-year relapse. KIR mismatching was not associated with NRM, acute or chronic GVHD.Conclusions: Haplo-identical transplant with PTCy and PBSC graft is associated with favorable outcome in patients with acute leukemia. KIR ligand mismatching may be associated with increased relapse rate and worse survival in these patients. Donors with KIR mismatching should be avoided when possible in the PBSC haplo-identical setting. [Display omitted] DisclosuresCiceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Mohty:Sanofi: Honoraria, Speakers Bureau.

A noninvasive diagnostic approach to retrospective donor HLA typing in kidney transplant patients using urine.

Antibody-mediated rejection (AMR) is a major obstacle to long-term kidney transplantation. AMR is mostly caused by donor specific HLA antibodies, which can arise before or any time after transplantation. Incomplete donor HLA typing and unavailability of donor DNA regularly preclude the assessment of donor-specificity of circulating anti-HLA antibodies. In our centre, this problem arises in approximately 20% of all post-transplant HLA-antibody assessments. We demonstrate that this diagnostic challenge can be resolved by establishing donor renal tubular cell cultures from recipient´s urine as a source of high-quality donor DNA. DNA was then verified for genetic origin and purity by fluorescence insitu hybridization and short tandem repeat analysis. Two representative cases highlight the diagnostic value of this approach which is corroborated by analysis of ten additional patients. The latter were randomly sampled from routine clinical care patients with available donor DNA as controls. In all 12 cases, we were able to perform full HLA typing of the respective donors confirmed by cross-comparison to results from the stored 10 donor DNAs. We propose that this noninvasive diagnostic approach for HLA typing in kidney transplant patients is valuable to determine donor specificity of HLA antibodies, which is important in clinical assessment of suspected AMR.

Four novel HLA class II alleles identified using next-generation sequencing.

We describe here four novel HLA class II alleles, DPA1*01:33, DPB1*1082:01, DPB1*1144:01, and DQA1*01:45, identified in our laboratory following the implementation of next generation sequencing (NGS) for routine high resolution HLA typing of donors and recipients for solid organ transplant and hematopoetic stem cell transplant. The NGS technology has improved our ability to match at all clinically HLA loci and to facilitate the interpretation of allele specific antibody and eplet matching.

MO1001THE OUTCOME OF PLASMA CELL-RICH ACUTE REJECTION IN KIDNEY TRANSPLANTATION, IS IT REALLY POOR?

Abstract Background and Aims The outcome of Plasma cell-rich acute rejection (PCAR) in kidney transplant is reported to be poor. However, PCAR which can be associated with any type of rejection, may not be considered as independent morphological prognostic feature. Different treatment modalities were prescribed with variable responses. We report here four cases of PCAR and describe their presentations, type of rejection, associated conditions and treatment outcome. Method Out of 1920 kidney transplant recipients under follow up in our centre from 1996 till 2019, four patients were reported to have PCAR according to 2007 Banff classification. They were re-evaluated based on 2015 Banff classification. The treatment protocol was tailored according to the type of rejection and associated conditions. Results The four patients, aged 28, 44, 46 and 54 years, had live unrelated renal transplant done somewhere abroad with no data about donor HLA typing. Two of them were females. One had high PRA and she was positive for HBsAg. One patient received induction immunosuppression with basiliximab. They all received prednisolone, mycophenolate and cyclosporine as the maintenance immunosuppression and had immediate graft function. Rejection happened between 23 to 180 months post-transplant. Two patients had acute T-cell mediated Banff 1A rejections with features consistent with early membranous nephropathy. One had acute T-cell mediated rejection Banff 1B and the fourth had borderline T-cell mediated rejection with morphological changes suggestive of chronic active antibody mediated rejection (AMR). Plasma cells constituted 10 to 30% of the interstitial infiltration. All patients received solumedrol pulse. Both patients with features of membranous nephropathy received rituximab and one of them had additionally IVIG. The patient with AMR received plasma exchange and IVIG. However, she did not receive rituximab as she was positive for HBsAg. All patients responded well to treatment and the mean improvement in eGFR was 12.8%, 24.9%, 40.3% and 39.1% at 1-, 3-, 6- and 12-months post treatment. Repeat kidney biopsy at 3 to 12 weeks post treatment showed resolution of plasma cell infiltration in all patients. Conclusion Outcome of PCAR management was favourable among our patients irrespective of the type of rejection and associated conditions.