57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Univ.-Prof. Dr. rer. nat.

Vorsitz

Prof. Dr. med.

Dr. med.

Prof.

Prof. Dr.

Eplet mismatch scores and development of donor specific antibodies in a German kidney transplantation cohort: are there high-risk eplets?

Immunogenetics and Basic Immunology

Eplet Mismatch Scores in einer deutschen Kohorte von Nierentransplantierten: Gibt es high-risk eplets?

High-resolution typing of HLA loci in the setting of solid organ transplantation allows for the assessment of the so-called eplet mismatch (EMM) score between donor and recipient. Eplets are key elements of epitopes, consisting of superficially located amino acid residues and represent the actual target structures of HLA antibodies. Our aim was to unravel whether there are high-risk EMM that are more likely to lead to the formation of de novo donorspecific antibodies (dnDSA) than others.We examined 148 kidney donor-recipient pairs regarding their mismatched eplets leading to the development of dnDSA, transplanted at the Goethe university hospital Frankfurt between 2018 and 2022. HLA high-resolution typing of donor and patient for 11 HLA loci was carried out using NGSgo-MX11-3 kit (GenDx) and MiSeq Sequencer (Illumina). Eplet analysis was performed using HLA Matchmaker algorithm (HLA Fusion v.4.4.0, OneLambda-ThermoFisher). Only confirmed HLA eplets were considered. Monitoring for HLA class I and class II antibodies was performed by single antigen microbead assays (SAB) (OneLambda- Lab Screen and Lifecodes-Immucor) for screening and/or for antibody identification. Graph Pad Prism was used for student´s t-test.In our cohort, 39 patients developed dnDSA, 4 of them suffered from graft failure. The medium EMM loads for class I and II of patients with dnDSA were higher than in the group without (class I: 9.2±5.9 vs. 12±7.3 (mean±SD), p=0.04/class II: 10±5.7 vs. 15±7.2, p=0.001). The mean MM load for both the DRB1 and the DQB1 locus alone was 6 each (range: 1-13/2-12) for patients with dnDSA, the majority of the values were below thresholds proposed earlier (Wiebe et al., 2017). Regarding the EMM there were some which led more often to the formation of dnDSA than others: 16 of the 146 eplets were involved in dnDSA formation with a probability of ≥ 25% (90th percentile) when they occurred. An accumulation of such EMMs was observed in the DQ locus.We confirmed that some EMM lead more frequently to the formation of dnDSA. Some of our high-risk eplets (163R, 104A, 57V) were associated with increased risk of DCGF in a large cohort with imputed high-resolution typing (Mohammadhassanzadeh et al., 2021). EMM thresholds allow basic risk-stratification but thorough characterization of individual EMMs enable comprehensive assessment of the immunogenicity of a transplant and might provide improved matching strategies with reduced dnDSA formation.References:Mohammadhassanzadeh, H., Oualkacha, K., Zhang, W., Klement, W., Bourdiec, A., Lamsatfi, J., Yi, Y., Foster, B., Keown, P., Gebel, H. M., Claas, F., Sapir-Pichhadze, R., &amp; Genome Canada Transplant, C. (2021). On Path to Informing Hierarchy of Eplet Mismatches as Determinants of Kidney Transplant Loss. Kidney Int Rep, 6(6), 1567-1579. https://doi.org/10.1016/j.ekir.2021.03.877Wiebe, C., Rush, D. N., Nevins, T. E., Birk, P. E., Blydt-Hansen, T., Gibson, I. W., Goldberg, A., Ho, J., Karpinski, M., Pochinco, D., Sharma, A., Storsley, L., Matas, A. J., &amp; Nickerson, P. W. (2017). Class II Eplet Mismatch Modulates Tacrolimus Trough Levels Required to Prevent Donor-Specific Antibody Development. J Am Soc Nephrol, 28(11), 3353-3362. https://doi.org/10.1681/ASN.2017030287There are no conflicts of interests.

Poster lecture

PD Dr.

PD Dr. rer. nat.

Early genotype-based dose adjustment of tacrolimus reduces risk for de novo donor-specific HLA antibodies and rejection in CYP3A5 expressers among renal transplant patients

Organ Transplatation

Frühe Genotypen-basierte Tracrolimus Dosierung reduziert das Risiko von de novo Donor-spezifischen HLA Antikörpern und Abstoßungen bei CYP3A5 Expressoren nach Nierentransplantation

The single nucleotide polymorphism rs776746 in the CYP3A5 gene slows down metabolism of tacrolimus via CYP3A5 enzyme. Our previous retrospective single-center cohort study showed a trend towards low tacrolimus trough levels and an increased risk for development of de novo donor-specific anti-HLA antibodies (DSA) and antibody-mediated rejection in CYP3A5 expressers carrying at least one copy of the wild-type allele at a follow-up of 3 years. We hypothesized that detection of CYP3A5 expresser status immediately post renal transplant, allowing early genotype-based dose adjustment of tacrolimus, might prevent the occurrence of de novo DSA and ABMR and improve renal transplant outcome.A total of 160 renal allograft recipients who underwent renal transplantation between May 2019 and 2022 were genotyped for CYP3A5 rs776746 polymorphism within the first two weeks post-transplant, and consequently genotype-based dose adjustment of tacrolimus was performed. The CYP3A5 genotype was determined by Pyrosequencing. After determination of CYP3A5 expresser status, patients were followed up for 2 years and development of calcineurin inhibitor (CNI) nephrotoxicity, rejections and de novo DSA was retrospectively analyzed.CYP3A5 expresser status was detected in 33 (21%) patients, whereas 127 (79%) patients were non-expressers. Tacrolimus trough levels were similar in CYP3A5 expressers and non-expressers over the follow-up period of 2 years. However, we observed a trend towards slightly increased tacrolimus trough levels in CYP3A5 expressers, who as expected required tacrolimus dosages twice as high as non-expressers. Correspondingly, CYP3A5 expressers had significantly lower concentration-to-dose ratios compared to non-expressers at all indicated time points. CNI nephrotoxicity-free survival rates were comparable between CYP3A5 expressers and non-expressers (p=0.49). Rejection-free survival rates (p=0.89) and de novo anti-HLA antibody-free survival rates (p=0.57) also did not differ between the two groups. The frequency of de novo DSA (2 (6%) vs. 5 (4%), p=0.6) and de novo DSA-free survival (p=0.61) were similar in CYP3A5 expressers and non-expressers.Early detection of CYP3A5 expresser status resulting in genotype-based adaption of tacrolimus dosage was not associated with increased incidence of CNI toxicity in CYP3A5 expressers but protected them from developement of transplant rejection and de novo DSA formation.K.S. has no conflict of interest.

PD Dr. Dr.

Immunotherapies beyond cancer: repurposing sibrotuzumab and NK-cells as a synergistic approach to treat fibrosis

Immunotherapy and Gene Therapy

Immuntherapien außerhalb der Onkologie: Repurposing von Sibrotuzumab und NK-Zellen als synergistischer Ansatz zur Behandlung von Fibrose

Fibrosis occurs in almost all chronic (inflammatory) diseases such as heart failure, (pulmonary) hypertension, systemic sclerosis, fatty liver disease and graft-versus-host disease. The uncontrolled secretion of extracellular matrix and inflammatory cytokines by dysregulated fibroblasts leads to a gradual loss of tissue function and contributes to 45% of deaths in the western world. Despite a clear medical need, effective antifibrotic therapies are lacking. However, fibroblast activation protein alpha (FAPα) has emerged as a potential fibroblast-specific target. Here, we present a novel strategy using the therapeutic FAPα antibody sibrotuzumab in combination with high affinity Fc receptor expressing NK92 cells (FCR-NK92) to eliminate FAPα+ fibroblasts and treat fibrosis.In the present study, human cardiac (HAF-SRK01), pulmonary (MRC5) and dermal (HFF1) fibroblasts and FCR-NK92 ± sibrotuzumab were used in flow cytometry, cytotoxicity and western blot experiments to assess the antifibrotic potential of NK-mediated antibody-dependent cellular cytotoxicity.First, we demonstrated specific activation of sibrotuzumab-loaded FCR-NK92 in the presence of human recombinant FAPα as determined by CD107A expression in a flowcytometric degranulation assay. Specific fibroblast killing was determined via europium release assay indicating significant fibroblast lysis after 2h. As therapeutic interventions targeting fibroblasts should be cell-specific to avoid off-target effects, we used a heterocellular culture model (fibroblasts and NK-resistant non-fibroblasts) to characterize the extent of specific fibroblast killing. After 4h, the FAPα+ fibroblast population was reduced by approximately 50% in the sibrotuzumab-treated cultures, whereas the non-fibroblast population (FAPα-) remained unchanged. To evaluate a functional antifibrotic effect, we measured protein expression of collagen 1 by western blot. After overnight co-culture, collagen 1 expression was abolished in all samples treated with FCR-NK92 and sibrotuzumab compared to control.As of now, antifibrotic cellular immunotherapies remain a largely untapped therapeutic opportunity. The results of our study highlight the promising potential of targeted cellular immunotherapy for the treatment of fibrosis, offering a compelling rationale for further exploration of this approach in the management of debilitating fibrotic diseases.The authors declare no conflict of interest.

Plasma transfusion from blood donors with high neutralizyting capacity against human cytomegalovirus (HCMV) increases the neutralization capacity against HCMV in hematopoietic stem cell recipients

Plasmatransfusion von Spendern mit hoher Neutralisationskapazität gegen humanes Zytomegalievirus (HCMV) erhöht bei stammzelltransplantierten Patienten die Neutralisationskapazität gegen HCMV

Immunoglobulins have been repeatedly applied to treat human cytomegalovirus (HCMV) infections, but without clear evidence of clinical efficacy. One explanation for these disappointing results is the limited neutralizing potency of the immunoglobulins used. An increase in the neutralizing antibody titers of the treated patients has also not yet been demonstrated.In a Phase I clinical trial, we tested whether the transfer of plasma from previously identified blood donors who neutralize HCMV exceptionally well (HCMV elite neutralizers) can increase the neutralization capacity in the serum of treated patients.The study included 12 stem cell transplant recipients who were HCMV seronegative and thus at risk for HCMV reactivation and disease. Patients received one or two plasma units of elite neutralizers in each of four treatment weeks. Serum samples were collected from patients before and after treatment and on days 2 and 4 of the treatment week. As a primary endpoint, neutralization titers in patients' serum samples were determined with an immunofluorescence-based assay using HCMV strain TB40/E, resulting in time curves of neutralization titers over the entire treatment period. Correlation analyses were performed to investigate whether the increase achieved by the treatment correlates with the neutralization capacity of the transferred plasma units. To analyze strain-specific differences, samples from before and after treatment were re-evaluated with seven laboratory strains representing almost all glycoprotein genotypes.On average, the transfused plasma units had an approximately 10-fold higher mean neutralizing capacity than the patients' sera. The combined curves of all patients showed that the plasma transfusion increased the neutralization capacity of the patients, on average by 83% immediately after transfusion and by 33% after one week. Over a period of four weeks, the cumulative increase in neutralization titer was 132%. The increase achieved in individual patients was highly variable and correlated with low baseline titers of the patients and high titers of the transferred plasma.Adoptive transfer of anti-HCMV antibodies can increase neutralization titers in a relevant patient group. Patients with a low neutralization capacity may benefit from the transfer of neutralizing antibodies, whereas patients with a high neutralization capacity will probably not.None

Gene-specific knockouts to improve the proliferation and persistence of NK cells in immunotherapy

Spezifische Gene knockouts zur Verbesserung der Proliferation und Persistenz von NK Zellen in der Immuntherapie

Natural killer (NK) cells are potent cytotoxic innate lymphocytes, and the potential of NK cell therapy to treat cancer is currently explored by many clinical trials. However, one of the main drawbacks of such approaches is the limited in vitro expansion capacity of NK cells and, upon transfusion, their insufficient in vivo persistence.We performed RNA-Seq analysis on NK cells expanded in vitro to determine gene expression changes driven by IL2 and IL15. CRISPR/Cas9-mediated genome editing was used to knockout genes negatively controlling the proliferation and restricting the expansion potential of NK cells.Primary NK cells were expanded by a factor 500 in vitro using standard pre-clinical protocols. We identified that IL2 and IL15 signaling in these cells involved the induction of negative regulators such as SOCS family genes to limit the proliferation. While meaningful for regular immune reactions, the therapeutic potential of NK cells could be improved by the elimination of inhibitory factors. Thus, we efficiently knocked out CISH, SOCS1 and SOCS2 as shown on the genomic as well as on the protein level. Indeed, these knockout cells had a competitive advantage over unmodified NK cells.Genome editing of NK cells can be used to increase the proliferative capacity of NK cells. This technical advance will be extended and allow the derivation of NK cells with strongly improved persistence and efficacy in adoptive immunotherapy.no disclosures

Haplotype phasing by long read nanopore sequencing of the CR1 gene coding for the Knops blood group system

Immunohematology

Haplotyp Identifikation mittels Long Read Sequenzierung des CR1 Gens für das Knops Blutgruppensystem

The antigens of the Knops blood group system (KN; ISBT 022) are located on the complement receptor 1 (CR1). All KN antigens identified so far are located in the Long Homologous Repeats LHR-C and LHR-D. Massively parallel sequencing (MPS) of all 39 exons of the CR1 gene in patients with suspected alloantibodies to KN antigens revealed several CR1 variants of unknown significance (VUS). The variants were located in different CR1 exons and phasing of the haplotypes remained unknown. We established long read Oxford Nanopore Technologies (ONT) sequencing for the LHR-C and -D region of CR1 for haplotype phasing.The CR1 LHR-C and -D region was amplified by long range PCR with 5 overlapping PCR products from genomic DNA samples of 10 patients with at least 2 distant VUS. ONT sequencing was performed on MinION with R10.4.1 flow cells (Oxford Nanopore Technologies, Oxford, UK). After base calling and filtering the sequence data were aligned to the chromosme 1 sequence of the human reference genome GRCh38.p14 by using the Geneious Prime software (Version 2023.2.1; Biomatters Inc., Boston, MA, USA).PCR amplification of the LHR-C and -D region was achieved from all samples with amplicon sizes of 10,601 to 12,797 bps. Long range ONT sequencing of the large amplicons confirmed the variants in the exons previously identified by MPS. Further variants were identified in the overlapping amplicon regions and were used for haplotype phasing of distant exon variants. In 6 samples we were able to identify the CR1 haplotypes including exon and intron variants with a distance of up to 41,699 bps. E.g., haplotype 1: c.3623A (exon 22, p.1208His, KN11) – c.4843A (exon 29, p.1615Ile, KN9) – c.5480C (exon 33, VUS); haplotype 2: c.3623G (exon 22, p.1208Arg, KN12) – c.4843G (exon 29, p.1615Val, KN10) – c.5480G (exon 33, VUS).Long read ONT sequencing of large PCR products is suitable for haplotype phasing of the CR1 gene. The knowledge of the haplotypes could be useful especially in cases with alloantibodies against an unknown KN antigen and two ore more VUS in CR1. We were able to allocate 4 VUS to haplotypes of the LHR-C and -D region.no disclosures

Investigation of antibody-induced Fc-gamma-RIIA-mediated procoagulant platelet formation in real-time

Hemostaseology

Untersuchung der Antikörper-vermittelten Formation von prokoagulanten Plättchen in Echtzeit

Heparin-induced thrombocytopenia is a prothrombotic disease characterized by a rapid drop in platelet (PLT) count and high risk for the onset of thromboembolic events. Anti-platelet factor 4/heparin immunoglobulin G (IgG) immune complexes that activate PLTs and different immune cells are the causing factor for HIT. Although PLTs are well characterized for being pivotal in the complex pathophysiology of HIT, a deeper knowledge regarding the kinetics of antibody (Ab)-induced Fc-gamma-RIIA-mediated PLT alterations is missing. Utilizing a flow cytometry (FC)-based kinetic protocol and a simplified in vitro model of HIT, we aimed to investigate Ab-induced PLT alterations in real time.PLTs from healthy individuals were loaded with the calcium-indicator Fluo-5N which was followed by dual agonist stimulation with thrombin (THR; [0.125 U/mL]) and convulxin (CVX, agonist of collagen GPVI receptor; [500 ng/mL]) or with monoclonal antibody (moAb) anti-CD9 or isotype control (Iso; [both 5 µg/mL]) for 15 min under continuous measurement in FC. Changes in PLTs phenotype were assessed by co-staining with PE-labelled moAb PAC-1 and Annexin-V (AnxV)-Cy5, detecting the activated fibrinogen receptor and procoagulant PLT phosphatidylserine (PS), respectively.Compared to control, stimulation of PLTs with anti-CD9 resulted in the formation of procoagulant PLTs that were characterized by a high F5N signal, increased binding of AnxV and low binding of PAC-1. Although dual agonist stimulation with THR+CVX resulted in significant higher formation of F5N positive PLTs (mean %±SEM: 33.6±2.8 vs. 15.2±1.2), higher binding of AnxV was detected following stimulation with anti-CD9 (mean fold increase of AnxV binding±SEM: 25.23±1.4 vs. 3.7±0.5, p&lt;0.0001). Most importantly, while the presence of the direct thrombin inhibitor hirudin did not show the potential to prevent anti-CD9-induced procoagulant PLT formation, specific FcγRIIA blockade using moAb IV.3 resulted in a nearly complete abrogation of Ab-induced procoagulant PLT formation (mean %±SEM: 15.2±1.2 vs. 0.80±0.3).First findings from our kinetic studies indicate that anti-CD9 induces procoagulant PLTs in a FcγRIIA-dependent manner. Future studies with this time resolved model of Ab-induced PLT activation will allow the identification of the critical events that are essential for the generation of Ab-induced procoagulant PLTs in HIT.The authors have no conflicts of interest to disclose.

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