Publications by authors named "Rainer Blasczyk"

172 Publications

Prolonged storage of purified granulocyte concentrates: Introduction of a new purification method.

Transfusion 2021 Nov 16. Epub 2021 Nov 16.

Artcline GmbH, Rostock, Germany.

Background: Use of donor granulocyte concentrate (GC) has been limited due to its short storage time of 6-24 h, which is partially due to residual red blood cells (RBCs) and platelets and the resulting lactate production leading to an acidotic milieu. To increase this storage time, we developed a closed system procedure compatible with standard blood bank technologies to remove RBC and platelets and to enrich the GC.

Methods: Standard GCs (sGCs) were sedimented, washed twice with 0.9% sodium chloride (NaCl), and resuspended in blood group-identical fresh frozen plasma. The resulting purified GCs (pGCs) were then stored in platelet bags at a cell concentration of about 5 × 10  ± 1.8 × 10 leukocytes/ml without agitation at room temperature for up to 72 h. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst were monitored daily.

Results: A significant reduction in RBC (98%) through sedimentation, and platelets (96%) by washing, purified the white blood cell (WBC) population and enriched the granulocytes to 96% of the WBC in the pGC. After 72 h of storage, over 90% of the initial WBC count of pGC remained, was viable (≥97%), and the granulocytes exhibited a high phagocytosis and oxidative burst functionality, comparable to sGC after 24 h.

Conclusion: Purification extends the maximum storage period of GC from 24 to 72 h and may therefore improve the availability of GC and its clinical use.
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http://dx.doi.org/10.1111/trf.16732DOI Listing
November 2021

Genetic modification of limbs using ex vivo machine perfusion.

Hum Gene Ther 2021 Nov 13. Epub 2021 Nov 13.

Hannover Medical School, 9177, Institute of Transfusion Medicine and Transplant Engineering, Hannover, Niedersachsen, Germany;

Genetic engineering is a promising tool to repair genetic disorders, improve graft function or to reduce immune responses towards the allografts. Ex vivo organ perfusion systems have the potential to mitigate ischemic-reperfusion injury, prolong preservation time or even rescue organ function. We aim to combine both technologies to develop a modular platform allowing the genetic modification of vascularized composite (VC) allografts. Rat hind limbs were perfused ex vivo under subnormothermic conditions with lentiviral vectors. Specific perfusion conditions such as controlled pressure, temperature and flow rates were optimized to support the genetic modification of the limbs. Genetic modification was detected in vascular, muscular and dermal limb tissues. Remarkably, skin follicular and interfollicular keratinocytes as well as endothelial cells (ECs) showed stable transgene expression. Furthermore, levels of injury markers such as lactate, myoglobin and lactate dehydrogenase (LDH) as well as histological analyses showed that ex vivo limb perfusion with lentiviral vectors did not cause tissue damage and limb cytokine secretion signatures were not significantly affected. The use of ex vivo VC perfusion in combination with lentiviral vectors allows an efficient and stable genetic modification of limbs representing a robust platform to genetically engineer limbs towards increasing graft survival after transplantation.
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http://dx.doi.org/10.1089/hum.2021.199DOI Listing
November 2021

Generation of HLA Universal Megakaryocytes and Platelets by Genetic Engineering.

Front Immunol 2021 27;12:768458. Epub 2021 Oct 27.

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Germany.

Patelet transfusion refractoriness remains a relevant hurdle in the treatment of severe alloimmunized thrombocytopenic patients. Antibodies specific for the human leukocyte antigens (HLA) class I are considered the major immunological cause for PLT transfusion refractoriness. Due to the insufficient availability of HLA-matched PLTs, the development of new technologies is highly desirable to provide an adequate management of thrombocytopenia in immunized patients. Blood pharming is a promising strategy not only to generate an alternative to donor blood products, but it may offer the possibility to optimize the therapeutic effect of the produced blood cells by genetic modification. Recently, enormous technical advances in the field of production of megakaryocytes (MKs) and PLTs have been achieved by combining progresses made at different levels including identification of suitable cell sources, cell pharming technologies, bioreactors and application of genetic engineering tools. In particular, use of RNA interference, TALEN and CRISPR/Cas9 nucleases or nickases has allowed for the generation of HLA universal PLTs with the potential to survive under refractoriness conditions. Genetically engineered HLA-silenced MKs and PLTs were shown to be functional and to have the capability to survive cell- and antibody-mediated cytotoxicity using and models. This review is focused on the methods to generate genetically engineered MKs and PLTs with the capacity to evade allogeneic immune responses.
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http://dx.doi.org/10.3389/fimmu.2021.768458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8579098PMC
October 2021

Case Report: Convalescent Plasma Therapy Induced Anti-SARS-CoV-2 T Cell Expansion, NK Cell Maturation and Virus Clearance in a B Cell Deficient Patient After CD19 CAR T Cell Therapy.

Front Immunol 2021 12;12:721738. Epub 2021 Aug 12.

Institute of Immunology, Hannover Medical School, Hannover, Germany.

Here, we described the case of a B cell-deficient patient after CD19 CAR-T cell therapy for refractory B cell Non-Hodgkin Lymphoma with protracted coronavirus disease 2019 (COVID-19). For weeks, this patient only inefficiently contained the virus while convalescent plasma transfusion correlated with virus clearance. Interestingly, following convalescent plasma therapy natural killer cells matured and virus-specific T cells expanded, presumably allowing virus clearance and recovery from the disease. Our findings, thus, suggest that convalescent plasma therapy can activate cellular immune responses to clear SARS-CoV-2 infections. If confirmed in larger clinical studies, these data could be of general importance for the treatment of COVID-19 patients.
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http://dx.doi.org/10.3389/fimmu.2021.721738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387963PMC
September 2021

Donors for SARS-CoV-2 Convalescent Plasma for a Controlled Clinical Trial: Donor Characteristics, Content and Time Course of SARS-CoV-2 Neutralizing Antibodies.

Transfus Med Hemother 2021 May 21;48(3):137-147. Epub 2021 Apr 21.

Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen and University Hospital Ulm, Ulm, Germany.

Background: Convalescent plasma is one of the treatment options for COVID-19 which is currently being investigated in many clinical trials. Understanding of donor and product characteristics is important for optimization of convalescent plasma.

Methods: Patients who had recovered from CO-VID-19 were recruited as donors for COVID-19 convalescent plasma (CCP) for a randomized clinical trial of CCP for treatment of severe COVID-19 (CAPSID Trial). Titers of neutralizing antibodies were measured by a plaque-reduction neutralization test (PRNT). Correlation of antibody titers with host factors and evolution of neutralizing antibody titers over time in repeat donors were analysed.

Results: A series of 144 donors (41% females, 59% males; median age 40 years) underwent 319 plasmapheresis procedures providing a median collection volume of 850 mL and a mean number of 2.7 therapeutic units per plasmapheresis. The majority of donors had a mild or moderate course of COVID-19. The titers of neutralizing antibodies varied greatly between CCP donors (from <1:20 to >1:640). Donor factors (gender, age, ABO type, body weight) did not correlate significantly with the titer of neutralizing antibodies. We observed a significant positive correlation of neutralization titers with the number of reported COVID-19 symptoms and with the time from SARS-CoV-2 diagnosis to plasmapheresis. Neutralizing antibody levels were stable or increased over time in 58% of repeat CCP donors. Mean titers of neutralizing antibodies of first donation and last donation of repeat CCP donors did not differ significantly (1:86 at first compared to 1:87 at the last donation). There was a significant correlation of neutralizing antibodies measured by PRNT and anti-SARS-CoV-2 IgG and IgA antibodies which were measured by ELISA. CCP donations with an anti-SARS-CoV-2 IgG antibody content above the 25th percentile were substantially enriched for CCP donations with higher neutralizing antibody levels.

Conclusion: We demonstrate the feasibility of collection of a large number of CCP products under a harmonized protocol for a randomized clinical trial. Titers of neutralizing antibodies were stable or increased over time in a subgroup of repeat donors. A history of higher number of COVID-19 symptoms and higher levels of anti-SARS-CoV-2 IgG and IgA antibodies in immunoassays can preselect donations with higher neutralizing capacity.
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http://dx.doi.org/10.1159/000515610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216018PMC
May 2021

Humoral and cellular immune responses against SARS-CoV-2 variants and human coronaviruses after single BNT162b2 vaccination.

Clin Infect Dis 2021 Jun 16. Epub 2021 Jun 16.

Department of Rheumatology and Immunology, Hannover Medical School, Hannover, Germany.

Background: Vaccine-induced neutralizing antibodies are key in combating the COVID-19 pandemic. However, delays of boost immunization due to limited availability of vaccines may leave individuals vulnerable to infection and prolonged or severe disease courses. The emergence of SARS-CoV-2 variants of concern (VOC), B.1.1.7 (United Kingdom), B.1.351 (South Africa), and P.1 (Brazil), may exacerbate this issue, as the latter two are able to evade control by antibodies.

Methods: We assessed humoral and T cell responses against SARS-CoV-2 WT, VOC and endemic human coronaviruses (hCoV) that were induced after single and double vaccination with BNT162b2.

Results: Despite readily detectable IgG against the receptor-binding domain (RBD) of the SARS-CoV-2 S protein at day 14 after a single vaccination, inhibition of SARS-CoV-2 S-driven host cell entry was weak and particularly low for the B.1.351 variant. Frequencies of SARS-CoV-2 WT and VOC specific T cells were low in many vaccinees after application of a single dose and influenced by immunity against endemic hCoV. The second vaccination significantly boosted T cell frequencies reactive for WT, B.1.1.7 and B.1.351 variants.

Conclusion: These results call into question whether neutralizing antibodies significantly contribute to protection against COVID-19 upon single vaccination and suggest that cellular immunity is central for the early defenses against COVID-19.
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http://dx.doi.org/10.1093/cid/ciab555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384414PMC
June 2021

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Mol Ther Methods Clin Dev 2021 Jun 9;21:621-641. Epub 2021 Apr 9.

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, 30625 Hannover, Germany.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14 monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34 cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.
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http://dx.doi.org/10.1016/j.omtm.2021.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142053PMC
June 2021

Allogeneic BK Virus-Specific T-Cell Treatment in 2 Patients With Progressive Multifocal Leukoencephalopathy.

Neurol Neuroimmunol Neuroinflamm 2021 07 17;8(4). Epub 2021 May 17.

From the Department of Neurology (F.H., N. Möhn, M.S., G.H., T.S.), Hannover Medical School; Institute of Transfusion Medicine and Transplant Engineering (B.E.-V., R.B.), Hannover Medical School; Department of Pediatric Hematology and Oncology (B.M.-K.), Hannover Medical School; Hannover Medical School (B.M.-K.), Institute for Transfusion Medicine; Department of Respiratory Medicine (J.G.), Hannover Medical School; Department of Diagnostic and Interventional Neuroradiology (N. Mahmoudi, M.P.W.), Hannover Medical School, Hannover; Department of Neurology (N. Mahmoudi, K.P., M.P.W.), Carl Von Ossietzky University, Oldenburg; and Institute of Virology (O.A.), Medical Faculty, Heinrich-Heine University Düsseldorf, Germany.

Objective: Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating opportunistic infection of the brain caused by the ubiquitously distributed JC polyomavirus. There are no established treatment options to stop or slow down disease progression. In 2018, a case series of 3 patients suggested the efficacy of allogeneic BK virus-specific T-cell (BKV-CTL) transplantation.

Methods: Two patients, a bilaterally lung transplanted patient on continuous immunosuppressive medication since 17 years and a patient with dermatomyositis treated with glucocorticosteroids, developed definite PML according to AAN diagnostic criteria. We transplanted both patients with allogeneic BKV-CTL from partially human leukocyte antigen (HLA) compatible donors. Donor T cells had directly been produced from leukapheresis by the CliniMACS IFN-γ cytokine capture system. In contrast to the previous series, we identified suitable donors by HLA typing in a preexamined registry and administered 1 log level less cells.

Results: Both patients' symptoms improved significantly within weeks. During the follow-up, a decrease in viral load in the CSF and a regression of the brain MRI changes occurred. The transfer seemed to induce endogenous BK and JC virus-specific T cells in the host.

Conclusions: We demonstrate that this optimized allogeneic BKV-CTL treatment paradigm represents a promising, innovative therapeutic option for PML and should be investigated in larger, controlled clinical trials.

Classification Of Evidence: This study provides Class IV evidence that for patients with PML, allogeneic transplant of BKV-CTL improved symptoms, reduced MRI changes, and decreased viral load.
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http://dx.doi.org/10.1212/NXI.0000000000001020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130010PMC
July 2021

COVID-19 immune signatures reveal stable antiviral T cell function despite declining humoral responses.

Immunity 2021 02;54(2):340-354.e6

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Germany. Electronic address:

Cellular and humoral immunity to SARS-CoV-2 is critical to control primary infection and correlates with severity of disease. The role of SARS-CoV-2-specific T cell immunity, its relationship to antibodies, and pre-existing immunity against endemic coronaviruses (huCoV), which has been hypothesized to be protective, were investigated in 82 healthy donors (HDs), 204 recovered (RCs), and 92 active COVID-19 patients (ACs). ACs had high amounts of anti-SARS-CoV-2 nucleocapsid and spike IgG but lymphopenia and overall reduced antiviral T cell responses due to the inflammatory milieu, expression of inhibitory molecules (PD-1, Tim-3) as well as effector caspase-3, -7, and -8 activity in T cells. SARS-CoV-2-specific T cell immunity conferred by polyfunctional, mainly interferon-γ-secreting CD4 T cells remained stable throughout convalescence, whereas humoral responses declined. Immune responses toward huCoV in RCs with mild disease and strong cellular SARS-CoV-2 T cell reactivity imply a protective role of pre-existing immunity against huCoV.
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http://dx.doi.org/10.1016/j.immuni.2021.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871825PMC
February 2021

Immunogenetics of xenotransplantation.

Int J Immunogenet 2021 Apr 7;48(2):120-134. Epub 2021 Jan 7.

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Germany.

Xenotransplantation may become the highly desired solution to close the gap between the availability of donated organs and number of patients on the waiting list. In recent years, enormous progress has been made in the development of genetically engineered donor pigs. The introduced genetic modifications showed to be efficient in prolonging xenograft survival. In this review, we focus on the type of immune responses that may target xeno-organs after transplantation and promising immunogenetic modifications that show a beneficial effect in ameliorating or eliminating harmful xenogeneic immune responses. Increasing histocompatibility of xenografts by eliminating genetic discrepancies between species will pave their way into clinical application.
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http://dx.doi.org/10.1111/iji.12526DOI Listing
April 2021

Low serum neutralizing anti-SARS-CoV-2 S antibody levels in mildly affected COVID-19 convalescent patients revealed by two different detection methods.

Cell Mol Immunol 2021 04 2;18(4):936-944. Epub 2020 Nov 2.

Institute of Immunology, Hannover Medical School, Hannover, Germany.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.
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http://dx.doi.org/10.1038/s41423-020-00573-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604543PMC
April 2021

CAR-T cells and TRUCKs that recognize an EBNA-3C-derived epitope presented on HLA-B*35 control Epstein-Barr virus-associated lymphoproliferation.

J Immunother Cancer 2020 10;8(2)

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Niedersachsen, Germany

Background: Immunosuppressive therapy or T-cell depletion in transplant patients can cause uncontrolled growth of Epstein-Barr virus (EBV)-infected B cells resulting in post-transplant lymphoproliferative disease (PTLD). Current treatment options do not distinguish between healthy and malignant B cells and are thereby often limited by severe side effects in the already immunocompromised patients. To specifically target EBV-infected B cells, we developed a novel peptide-selective chimeric antigen receptor (CAR) based on the monoclonal antibody TÜ165 which recognizes an Epstein-Barr nuclear antigen (EBNA)-3C-derived peptide in HLA-B*35 context in a T-cell receptor (TCR)-like manner. In order to attract additional immune cells to proximity of PTLD cells, based on the TÜ165 CAR, we moreover generated T cells redirected for universal cytokine-mediated killing (TRUCKs), which induce interleukin (IL)-12 release on target contact.

Methods: TÜ165-based CAR-T cells (CAR-Ts) and TRUCKs with inducible IL-12 expression in an all-in-one construct were generated. Functionality of the engineered cells was assessed in co-cultures with EBNA-3C-peptide-loaded, HLA-B*35-expressing K562 cells and EBV-infected B cells as PTLD model. IL-12, secreted by TRUCKs on target contact, was further tested for its chemoattractive and activating potential towards monocytes and natural killer (NK) cells.

Results: After co-cultivation with EBV target cells, TÜ165 CAR-Ts and TRUCKs showed an increased activation marker expression (CD137, CD25) and release of proinflammatory cytokines (interferon-γ and tumor necrosis factor-α). Moreover, TÜ165 CAR-Ts and TRUCKs released apoptosis-inducing mediators (granzyme B and perforin) and were capable to specifically lyse EBV-positive target cells. Live cell imaging revealed a specific attraction of TÜ165 CAR-Ts around EBNA-3C-peptide-loaded target cells. Of note, TÜ165 TRUCKs with inducible IL-12 showed highly improved effector functions and additionally led to recruitment of monocyte and NK cell lines.

Conclusions: Our results demonstrate that TÜ165 CAR-Ts recognize EBV peptide/HLA complexes in a TCR-like manner and thereby allow for recognizing an intracellular EBV target. TÜ165 TRUCKs equipped with inducible IL-12 expression responded even more effectively and released IL-12 recruited additional immune cells which are generally missing in proximity of lymphoproliferation in immunocompromised PTLD patients. This suggests a new and promising strategy to specifically target EBV-infected cells while sparing and mobilizing healthy immune cells and thereby enable control of EBV-associated lymphoproliferation.
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http://dx.doi.org/10.1136/jitc-2020-000736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604878PMC
October 2020

The Loss of HLA-F/KIR3DS1 Ligation Is Mediated by Hemoglobin Peptides.

Int J Mol Sci 2020 Oct 28;21(21). Epub 2020 Oct 28.

Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

The human leukocyte antigen (HLA)-Ib molecule, HLA-F, is known as a CD4 T-cell protein and mediator of HIV progression. While HLA-Ia molecules do not have the chance to select and present viral peptides for immune recognition due to protein downregulation, HLA-F is upregulated. Post HIV infection, HLA-F loses the affinity to its activating receptor KIR3DS1 on NK cells leading to progression of the HIV infection. Several studies aimed to solve the question of the biophysical interface between HLA ligands and their cognate receptors. It became clear that even an invariant HLA molecule can be structurally modified by the variability of the bound peptide. We recently discovered the ability of HLA-F to select and present peptides and the HLA-F allele-specific peptide selection from the proteomic content using soluble HLA (sHLA) technology and a sophisticated MS method. We established recombinant K562 cells that express membrane-bound HLA-F*01:01, 01:03 or 01:04 complexes. While a recombinant soluble form of KIR3DS1 did not bind to the peptide-HLA-F complexes, acid elution of the peptides resulted in the presentation of HLA-F open conformers, and the binding of the soluble KIR3DS1 receptor increased. We used CD4/HIV and CD4/HIV cells and performed an MS proteome analysis. We could detect hemoglobin as significantly upregulated in CD4 T-cells post HIV infection. The expression of cellular hemoglobin in nonerythroid cells has been described, yet HLA-Ib presentation of hemoglobin-derived peptides is novel. Peptide sequence analysis from HLA-F allelic variants featured hemoglobin peptides as dominant and shared. The reciprocal experiment of binding hemoglobin peptide fractions to the HLA-F open conformers resulted in significantly diminished receptor recognition. These results underpin the molecular involvement of HLA-F and its designated peptide ligand in HIV immune escape.
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http://dx.doi.org/10.3390/ijms21218012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672607PMC
October 2020

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes.

Int J Mol Sci 2020 Oct 16;21(20). Epub 2020 Oct 16.

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, 30625 Hannover, Germany.

Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.
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http://dx.doi.org/10.3390/ijms21207654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589913PMC
October 2020

CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease.

Mol Ther Oncolytics 2020 Sep 8;18:504-524. Epub 2020 Aug 8.

Laboratory of Regenerative Immune Therapies Applied, Hannover Medical School, 30625 Hannover, Germany.

Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3ζ signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350 293T cells . The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV B95-8 cell line, showing selectivity against gp350 cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34 cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV lymphoproliferation and lymphomagenesis.
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http://dx.doi.org/10.1016/j.omto.2020.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479496PMC
September 2020

Isolation, Cryopreservation, and Characterization of iPSC-Derived Megakaryocytes.

Methods Mol Biol 2021 ;2180:539-554

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Germany.

Current research in the field of transfusion medicine is focused on developing innovative approaches to generate populations of functional megakaryocytes (MKs) ex vivo. This may open perspectives to establish alternative therapies for donor platelet transfusion in the management of thrombocytopenic patients and pave the way for novel regenerative approaches. Efficient cryopreservation techniques can provide the opportunity for long-term storage and accumulation of necessary amounts of MKs in a ready-to-use manner. However, in this case, besides the viability, it is crucial to consider the recovery of functional MK properties after the impact of freezing. In this chapter, the possibility to cryopreserve iPSC-derived MKs is described. In particular, the methods for a comprehensive analysis of phenotypic and functional features of MKs after cryopreservation are proposed. The use of cryopreserved in vitro-produced MKs may benefit to the field of transfusion medicine to overcome the lack of sufficient blood donors.
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http://dx.doi.org/10.1007/978-1-0716-0783-1_27DOI Listing
March 2021

Reappearance of effector T cells is associated with recovery from COVID-19.

EBioMedicine 2020 Jul 7;57:102885. Epub 2020 Jul 7.

Institute of Immunology, Hannover Medical School, Germany; Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Germany. Electronic address:

Background: Elucidating the role of T cell responses in COVID-19 is of utmost importance to understand the clearance of SARS-CoV-2 infection.

Methods: 30 hospitalized COVID-19 patients and 60 age- and gender-matched healthy controls (HC) participated in this study. We used two comprehensive 11-colour flow cytometric panels conforming to Good Laboratory Practice and approved for clinical diagnostics.

Findings: Absolute numbers of lymphocyte subsets were differentially decreased in COVID-19 patients according to clinical severity. In severe disease (SD) patients, all lymphocyte subsets were reduced, whilst in mild disease (MD) NK, NKT and γδ T cells were at the level of HC. Additionally, we provide evidence of T cell activation in MD but not SD, when compared to HC. Follow up samples revealed a marked increase in effector T cells and memory subsets in convalescing but not in non-convalescing patients.

Interpretation: Our data suggest that activation and expansion of innate and adaptive lymphocytes play a major role in COVID-19. Additionally, recovery is associated with formation of T cell memory as suggested by the missing formation of effector and central memory T cells in SD but not in MD. Understanding T cell-responses in the context of clinical severity might serve as foundation to overcome the lack of effective anti-viral immune response in severely affected COVID-19 patients and can offer prognostic value as biomarker for disease outcome and control.

Funding: Funded by State of Lower Saxony grant 14-76,103-184CORONA-11/20 and German Research Foundation, Excellence Strategy - EXC2155"RESIST"-Project ID39087428, and DFG-SFB900/3-Project ID158989968, grants SFB900-B3, SFB900-B8.
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http://dx.doi.org/10.1016/j.ebiom.2020.102885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338277PMC
July 2020

NKG2A/CD94 Is a New Immune Receptor for HLA-G and Distinguishes Amino Acid Differences in the HLA-G Heavy Chain.

Int J Mol Sci 2020 Jun 19;21(12). Epub 2020 Jun 19.

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, 30625 Hannover, Germany.

Natural killer (NK) cell therapies are a tool to antagonize a dysfunctional immune system. NK cells recognize malignant cells, traffic to a tumor location, and infiltrate the solid tumor. The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G. To verify the results, we used the reciprocal method by expressing recombinant soluble heterodimeric NKG2A/CD94 molecules and used them to target HLA-G*01:01 expressing cells. NKG2A/CD94 could be confirmed as an immune receptor of HLA-G*01:01. Despite HLA-G is marginal polymorphic, we could previously demonstrate that the most common allelic subtypes HLA-G*01:01/01:03 and 01:04 differ in peptide repertoire, their engagement to NK cells, their catalyzation of dNK cell proliferation and their impact on NK cell development. Continuing these studies with regard to NKG2A/CD94 engagement we engineered recombinant single antigen presenting cells and targeted the surface expressed HLA-G*01:01, 01:03 or 01:04 molecules with NKG2A/CD94. Specificity and sensitivity of HLA-G*01:04/NKG2A/CD94 engagement could be significantly verified. The binding affinity decreases when using or cells as targets. These results demonstrate that the ligand-receptor assignment between HLA-G and NKG2A/CD94 is dependent of the amino acid composition in the HLA-G heavy chain. Understanding the biophysical basis of receptor-mediated events that lead to NK cell inhibition would help to remove non-tumor reactive cells and support personalized mild autologous NK cell therapies.
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http://dx.doi.org/10.3390/ijms21124362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352787PMC
June 2020

Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology.

Int J Mol Sci 2020 Jun 4;21(11). Epub 2020 Jun 4.

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, 30625 Hannover, Germany.

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing-thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-β1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing.
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http://dx.doi.org/10.3390/ijms21114029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312941PMC
June 2020

High-intensity interval training in allogeneic adoptive T-cell immunotherapy - a big HIT?

J Transl Med 2020 04 1;18(1):148. Epub 2020 Apr 1.

Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.

Background: Adoptive transfer of virus-specific T cells (VSTs) represents a prophylactic and curative approach for opportunistic viral infections and reactivations after transplantation. However, inadequate frequencies of circulating memory VSTs in the T-cell donor's peripheral blood often result in insufficient enrichment efficiency and purity of the final T-cell product, limiting the effectiveness of this approach.

Methods: This pilot study was designed as a cross-over trial and compared the effect of a single bout (30 min) of high-intensity interval training (HIT) with that of 30 min of continuous exercise (CONT) on the frequency and function of circulating donor VSTs. To this end, we used established immunoassays to examine the donors' cellular immune status, in particular, with respect to the frequency and specific characteristics of VSTs restricted against Cytomegalovirus (CMV)-, Epstein-Barr-Virus (EBV)- and Adenovirus (AdV)-derived antigens. T-cell function, phenotype, activation and proliferation were examined at different time points before and after exercise to identify the most suitable time for T-cell donation. The clinical applicability was determined by small-scale T-cell enrichment using interferon- (IFN-) γ cytokine secretion assay and virus-derived overlapping peptide pools.

Results: HIT proved to be the most effective exercise program with up to fivefold higher VST response. In general, donors with a moderate fitness level had higher starting and post-exercise frequencies of VSTs than highly fit donors, who showed significantly lower post-exercise increases in VST frequencies. Both exercise programs boosted the number of VSTs against less immunodominant antigens, specifically CMV (IE-1), EBV (EBNA-1) and AdV (Hexon, Penton), compared to VSTs against immunodominant antigens with higher memory T-cell frequencies.

Conclusion: This study demonstrates that exercise before T-cell donation has a beneficial effect on the donor's cellular immunity with respect to the proportion of circulating functionally active VSTs. We conclude that a single bout of HIT exercise 24 h before T-cell donation can significantly improve manufacturing of clinically applicable VSTs. This simple and economical adjuvant treatment proved to be especially efficient in enhancing virus-specific memory T cells with low precursor frequencies.
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http://dx.doi.org/10.1186/s12967-020-02301-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114817PMC
April 2020

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation.

J Cell Mol Med 2020 05 25;24(9):5070-5081. Epub 2020 Mar 25.

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)-silenced islet cell clusters (ICC). Single-cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA-silenced ICCs-derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs-derived cells. Xenogeneic T cell immune responses, NK cell and antibody-mediated cellular-dependent immune responses were significantly decreased in SLA-silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single-cell engineering and post-transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.
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http://dx.doi.org/10.1111/jcmm.15136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205796PMC
May 2020

Genetic Engineering of the Kidney to Permanently Silence MHC Transcripts During Organ Perfusion.

Front Immunol 2020 19;11:265. Epub 2020 Feb 19.

Hannover Medical School, Institute of Transfusion Medicine and Transplant Engineering, Hanover, Germany.

Organ gene therapy represents a promising tool to correct diseases or improve graft survival after transplantation. Polymorphic variation of the major histocompatibility complex (MHC) antigens remains a major obstacle to long-term graft survival after transplantation. Previously, we demonstrated that MHC-silenced cells are protected against allogeneic immune responses. We also showed the feasibility to silence MHC in the lung. Here, we aimed at the genetic engineering of the kidney toward permanent silencing of MHC antigens in a rat model. We constructed a sub-normothermic perfusion system to deliver lentiviral vectors encoding shRNAs targeting β2-microglobulin and the class II transactivator to the kidney. In addition, the vector contained the sequence for a secreted nanoluciferase. After kidney transplantation (ktx), we detected bioluminescence in the plasma and urine of recipients of an engineered kidney during the 6 weeks of post-transplant monitoring, indicating a stable transgene expression. Remarkably, transcript levels of β2-microglobulin and the class II transactivator were decreased by 70% in kidneys expressing specific shRNAs. Kidney genetic modification did not cause additional cell death compared to control kidneys after machine perfusion. Nevertheless, cytokine secretion signatures were altered during perfusion with lentiviral vectors as revealed by an increase in the secretion of IL-10, MIP-1α, MIP-2, IP-10, and EGF and a decrease in the levels of IL-12, IL-17, MCP-1, and IFN-γ. Biodistribution assays indicate that the localization of the vector was restricted to the graft. This study shows the potential to generate immunologically invisible kidneys showing great promise to support graft survival after transplantation and may contribute to reduce the burden of immunosuppression.
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http://dx.doi.org/10.3389/fimmu.2020.00265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042208PMC
March 2021

Variation in the Human Leukocyte Antigen system and risk for endemic Burkitt lymphoma in northern Uganda.

Br J Haematol 2020 05 18;189(3):489-499. Epub 2020 Feb 18.

Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Endemic Burkitt lymphoma (eBL) is an aggressive childhood B-cell lymphoma associated with Plasmodium falciparum (Pf) malaria and Epstein-Barr virus (EBV) infections. Variation in the Human Leukocyte Antigen (HLA) system is suspected to play a role, but assessments using less accurate serology-based HLA typing techniques in small studies yielded conflicting results. We studied 200 eBL cases and 400 controls aged 0-15 years enrolled in northern Uganda and typed by accurate high-resolution HLA sequencing methods. HLA results were analyzed at one- or two-field resolution. Odds ratios and 95% confidence intervals (aOR, 95% CI) for eBL risk associated with common HLA alleles versus alleles that were rare (<1%) or differed by <2% between the cases and controls as the reference category, were estimated using multiple logistic regression adjusting for age, sex, microgeography, region, malaria positivity and treatment history, and genetic variants associated with eBL. Compared to the controls, eBL cases had a lower frequency of HLA-A*02 (aOR = 0·59, 95% CI 0·38-0·91), HLA-B*41 (aOR = 0·36, 95% CI 0·13-1·00), and HLA-B*58 alleles (aOR = 0·59, 95% CI 0·36-0·97). eBL cases had a lower frequency of HLA-DPB1 homozygosity (aOR = 0·57, 95% CI 0·40-0·82) but a higher frequency of HLA-DQA1 homozygosity (aOR = 2·19, 95% CI 1·42-3·37). Our results suggest that variation in HLA may be associated with eBL risk.
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http://dx.doi.org/10.1111/bjh.16398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192769PMC
May 2020

Six-year experience with treatment of early donor-specific anti-HLA antibodies in pediatric lung transplantation using a human immunoglobulin-based protocol.

Pediatr Pulmonol 2020 03 7;55(3):754-764. Epub 2020 Jan 7.

Department of Cardiothoracic, Transplant and Vascular Surgery, Hannover Medical School, Hannover, Germany.

Objectives: Experience with the treatment of early donor-specific anti-HLA antibodies (eDSA) after lung transplantation in children is very limited. At our institution, we have treated patients with eDSA since 2013 with successive infusions of intravenous human immunoglobulins (IVIG), combined in some cases with a single dose of Rituximab and plasmapheresis (therapeutic plasma exchange [tPE]) or immunoabsorption. The aim of this study was to present the 6-year results of IVIG-based therapy in pediatric lung recipients.

Methods: Records of pediatric (<18 years old) patients transplanted at our institution between 01/2013 and 03/2019 were reviewed. Outcomes were compared between patients with eDSA treated with IVIG (IVIG group) and without eDSA (control group). Median (interquartile range [IQR]) follow-up amounted to 28 (12-52) months.

Results: During the study period, 66 lung-transplanted pediatric patients were included, of which 27 (41%) formed the IVIG group and 38 (57%) the control group. Among the IVIG patients, 14 (52%) patients showed concomitant graft dysfunction (possible clinical antibody-mediated rejection). The median time to eDSA detection was 24 (14-63) days after transplantation. eDSA were cleared in 25 (96%) of the 26 patients which completed treatment. At 3 years, graft survival (%) was 73 vs 85 (P = .65); freedom (%) from chronic lung allograft rejection (CLAD) was 89 vs 78 (P = .82); and from infection 47 vs 31 (P = .15), in IVIG vs control patients, respectively.

Conclusions: After lung transplantation, an IVIG-based treatment for eDSA yielded high eDSA clearance. IVIG and control patients showed similar CLAD-free and graft survival.
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http://dx.doi.org/10.1002/ppul.24639DOI Listing
March 2020

HLA-F Allele-Specific Peptide Restriction Represents an Exceptional Proteomic Footprint.

Int J Mol Sci 2019 Nov 8;20(22). Epub 2019 Nov 8.

Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

Peptide-dependent engagement between human leucocyte antigens class I (HLA-I) molecules and their cognate receptors has been extensively analyzed. HLA-F belongs to the non-classical HLA-Ib molecules with marginal polymorphic nature and tissue restricted distribution. The three common allelic variants HLA-F*01:01/01:03/01:04 are distinguished by polymorphism outside the peptide binding pockets (residue 50, α1 or residue 251, α3) and are therefore not considered relevant for attention. However, peptide selection and presentation undergoes a most elaborated extraction from the whole available proteome. It is known that HLA-F confers a beneficial effect on disease outcome during HIV-1 infections. The interaction with the NK cell receptor initiates an antiviral downstream immune response and lead to delayed disease progression. During the time of HIV infection, HLA-F expression is upregulated, while its interaction with KIR3DS1 is diminished. The non-polymorphic nature of HLA-F facilitates the conclusion that understanding HLA-F peptide selection and presentation is essential to a comprehensive understanding of this dynamic immune response. Utilizing soluble HLA technology we recovered stable pHLA-F*01:01, 01:03 and 01:04 complexes from K562 cells and analyzed the peptides presented. Utilizing a sophisticated LC-MS-method, we analyzed the complete proteome and matched the peptides presented by the respective HLA-F subtypes with detected proteins. All peptides featured a length of 8 to 24 amino acids and are not N-terminally anchored; the C-terminus is preferably anchored by Lys. To comprehend the alteration of the pHLA-F surface we structurally compared HLA-F variants bound to selected peptides. The peptides were selected from the same cellular content; however, no overlap between the proteomic source of F*01:01, 01:03 or 01:04 selected peptides could be observed. Recognizing the balance between HLA-F expression, HLA-F polymorphism and peptide selection will support to understand the role of HLA-F in viral pathogenesis.
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http://dx.doi.org/10.3390/ijms20225572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888383PMC
November 2019

Transfer of Hexon- and Penton-selected adenovirus-specific T cells for refractory adenovirus infection after haploidentical stem cell transplantation.

Transpl Infect Dis 2020 Feb 11;22(1):e13201. Epub 2019 Nov 11.

Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.

Adenovirus (HAdV) infections confer a high risk of morbidity and mortality for immunocompromised patients after stem cell transplantation (SCT). Treatment with standard antiviral drugs is of limited efficacy and associated with a high rate of adverse effects. HAdV-specific T cells are crucial for sustained viral elimination and the efficacy of adoptive T-cell therapy with donor-derived HAdV-specific T cells has been reported by several investigators. Here, we report our experience with the transfer of HAdV-specific T cells specific for penton, which was recently identified as an immunodominant target of T cells, and hexon in a 14-year-old boy after T-cell-depleted haploidentical SCT for myelodysplastic syndrome (MDS). He developed severe HAdV-associated enteritis complicated by acute graft-versus-host disease (GvHD). The patient received ten infusions of allogeneic HAdV-specific T cells manufactured from the haploidentical stem cell donor using the CliniMacs Interferon-γ (IFN-γ) cytokine capture and immunomagnetic selection. Initially, T cells were generated against the immunodominant target hexon and in subsequent transfers dual antigen-specific T cells against hexon and penton were applied. T-cell transfers were scheduled individually tailored to current immunosuppressive treatment. Each transfer was followed by reduction of HAdV load in peripheral blood and clinical improvement. Importantly, T-cell responses to both penton and hexon pools emerged in patient blood after repetitive transfers. Unfortunately, the patient experienced bacterial sepsis, and in this context, severe GvHD requiring intensive immunosuppression followed by secondary progression of HAdV infection. The patient succumbed to multiorgan failure 283 days after SCT. This case demonstrates the feasibility of HAdV-specific T-cell transfer even in the presence of immunosuppressive treatment. Targeting of multiple immunodominant viral proteins may prove valuable in patients with complicated HAdV infections.
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http://dx.doi.org/10.1111/tid.13201DOI Listing
February 2020

The Mechanistic Differences in HLA-Associated Carbamazepine Hypersensitivity.

Pharmaceutics 2019 Oct 15;11(10). Epub 2019 Oct 15.

Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

Drug hypersensitivity reactions that resemble acute immune reactions are linked to certain human leucocyte antigen (HLA) alleles. Severe and life-threatening Stevens Johnson Syndrome and Toxic Epidermal Necrolysis following treatment with the antiepileptic and psychotropic drug Carbamazepine are associated with HLA-B*15:02; whereas carriers of HLA-A*31:01 develop milder symptoms. It is not understood how these immunogenic differences emerge genotype-specific. For HLA-B*15:02 an altered peptide presentation has been described following exposure to the main metabolite of carbamazepine that is binding to certain amino acids in the F pocket of the HLA molecule. The difference in the molecular mechanism of these diseases has not been comprehensively analyzed, yet; and is addressed in this study. Soluble HLA-technology was utilized to examine peptide presentation of HLA-A*31:01 in presence and absence of carbamazepine and its main metabolite and to examine the mode of peptide loading. Proteome analysis of drug-treated and untreated cells was performed. Alterations in sA*31:01-presented peptides after treatment with carbamazepine revealed different half-life times of peptide-HLA- or peptide-drug-HLA complexes. Together with observed changes in the proteome elicited through carbamazepine or its metabolite these results illustrate the mechanistic differences in carbamazepine hypersensitivity for HLA-A*31:01 or B*15:02 patients and constitute the bridge between pharmacology and pharmacogenetics for personalized therapeutics.
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http://dx.doi.org/10.3390/pharmaceutics11100536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835980PMC
October 2019

Low immunogenic endothelial cells endothelialize the Left Ventricular Assist Device.

Sci Rep 2019 08 5;9(1):11318. Epub 2019 Aug 5.

SPP2014 - Towards an implantable lung, Hannover, Germany.

Low haemocompatibility of left ventricular assist devices (LVAD) surfaces necessitates anticoagulative therapy. Endothelial cell (EC) seeding can support haemocompatibility, however, the availability of autologous ECs is limited. In contrast, allogeneic ECs are readily available in sufficient quantity, but HLA disparities induce harmful immune responses causing EC loss. In this study, we investigated the feasibility of using allogeneic low immunogenic ECs to endothelialize LVAD sintered inflow cannulas (SIC). To reduce the immunogenicity of ECs, we applied an inducible lentiviral vector to deliver short-hairpins RNA to silence HLA class I expression. HLA class I expression on ECs was conditionally silenced by up to 70%. Sufficient and comparable endothelialization rates were achieved with HLA-expressing or HLA-silenced ECs. Cell proliferation was not impaired by cell-to-Sintered Inflow Cannulas (SIC) contact or by silencing HLA expression. The levels of endothelial phenotypic and thrombogenic markers or cytokine secretion profiles remained unaffected. HLA-silenced ECs-coated SIC exhibited reduced thrombogenicity. In contrast to native ECs, HLA-silenced ECs showed lower cell lysis rates when exposed to allogeneic T cells or specific anti-HLA antibodies. Allogeneic HLA-silenced ECs could potentially become a valuable source for LVAD endothelialization to reduce immunogenicity and correspondingly the need for anticoagulative therapy which can entail severe side effects.
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http://dx.doi.org/10.1038/s41598-019-47780-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683293PMC
August 2019

Battle between Host Immune Cellular Responses and HCMV Immune Evasion.

Int J Mol Sci 2019 Jul 24;20(15). Epub 2019 Jul 24.

Institute for Transfusion Medicine, Hannover Medical School, 30625 Hannover, Germany.

Human cytomegalovirus (HCMV) is ubiquitously prevalent. HCMV infection is typically asymptomatic and controlled by the immune system in healthy individuals, yet HCMV can be severely pathogenic for the fetus during pregnancy and in immunocompromised persons, such as transplant recipients or HIV infected patients. HCMV has co-evolved with the hosts, developed strategies to hide from immune effector cells and to successfully survive in the human organism. One strategy for evading or delaying the immune response is maintenance of the viral genome to establish the phase of latency. Furthermore, HCMV immune evasion involves the downregulation of human leukocyte antigens (HLA)-Ia molecules to hide infected cells from T-cell recognition. HCMV expresses several proteins that are described for downregulation of the HLA class I pathway via various mechanisms. Here, we review the wide range of immune evasion mechanisms of HCMV. Understanding the mechanisms of HCMV immune evasion will contribute to the development of new customized therapeutic strategies against the virus.
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http://dx.doi.org/10.3390/ijms20153626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695940PMC
July 2019
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