Publications by authors named "Britta Maecker-Kolhoff"

45 Publications

Primary post-transplant lymphoproliferative disorder of the central nervous system: characteristics, management and outcome in 25 paediatric patients.

Br J Haematol 2021 Mar 25. Epub 2021 Mar 25.

Department of Pediatric Hematology and Oncology, St. Anna Children's Hospital, Medical University of Vienna, Vienna, Austria.

Primary central nervous system (CNS) post-transplant lymphoproliferative disorder (PTLD) in childhood is rare. Twenty-five patients were retrieved from nine European Intergroup for Childhood Non-Hodgkin's Lymphoma and/or international Berlin-Frankfurt-Münster Study Group members. Types of allografts included kidney (n = 11), liver (n = 4), heart (n = 5), bowel (n = 1) and haematopoietic stem cells (n = 4). Eighteen were male, 16 ≥ 10 years old, 21 had monomorphic disease and 24 solid intracranial tumour masses. Four-year event-free and overall survival rates were 50% ± 10% and 74% ± 9% respectively. This report represents the largest paediatric series of CNS PTLD reported to date, showing favourable survival odds following systemic and intrathecal chemotherapy and rituximab administration.
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http://dx.doi.org/10.1111/bjh.17398DOI Listing
March 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

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

Matched Family Donor Lymphocyte Infusions as First Cellular Therapy for Patients with Severe Primary T Cell Deficiencies.

Transplant Cell Ther 2021 Jan 3;27(1):93.e1-93.e8. Epub 2020 Oct 3.

Department of Pediatrics, University Medical Center Ulm, Ulm, Germany.

Patients with primary immunodeficiencies caused by severe defects in T cell immunity are at risk of acquiring life-threatening infections. Cellular therapies are necessary to establish normal T cell function and to allow for long-term survival. This is most commonly achieved by hematopoietic stem cell transplantation (HSCT), but the outcome of this procedure is impaired if active infections are present at the time of HSCT. Donor lymphocyte infusions (DLIs) are a well-established therapeutic strategy following HSCT to treat viral infections, improve donor cell engraftment, or achieve graft-versus-leukemia activity in malignant disease. Here we present a cohort of 6 patients with primary T cell deficiencies who received transfusions of unselected mature donor lymphocytes prior and not directly related to allogeneic HSCT. DLIs obtained from the peripheral blood of HLA-identical (10/10) family donors were transfused without prior conditioning to treat or prevent life-threatening infections. All patients are alive with a follow-up of 0.5 to 16.5 years after the initial T cell administration. Additional cellular therapies were administered in 5 of 6 patients at 0.8 to 15 months after the first DLI. Mild cutaneous graft-versus-host disease (GVHD, stage ≤2) was observed in 3 of 6 patients and resolved spontaneously. We provide evidence that unselected HLA-identical DLIs can effectively prevent or contribute to overcome infections with a limited risk for GVHD in T cell deficient patients. The T cell system established by this readily available source can provide T cell function for years and can serve as a bridge to additional cellular therapies or, in specific conditions, as definite treatment.
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http://dx.doi.org/10.1016/j.bbmt.2020.09.037DOI Listing
January 2021

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

Plerixafor combined with standard regimens for hematopoietic stem cell mobilization in pediatric patients with solid tumors eligible for autologous transplants: two-arm phase I/II study (MOZAIC).

Bone Marrow Transplant 2020 09 3;55(9):1744-1753. Epub 2020 Mar 3.

IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

This study (NCT01288573) investigated plerixafor's safety and efficacy in children with cancer. Stage 1 investigated the dosage, pharmacokinetics (PK), pharmacodynamics (PD), and safety of plerixafor + standard mobilization (G-CSF ± chemotherapy). The stage 2 primary endpoint was successful mobilization (doubling of peripheral blood CD34+ cell count in the 24 h prior to first apheresis) in patients treated with plerixafor + standard mobilization vs. standard mobilization alone. In stage 1, three patients per age group (2-<6, 6-<12, and 12-<18 years) were treated at each dose level (160, 240, and 320 µg/kg). Based on PK and PD data, the dose proposed for stage 2 was 240 µg/kg (patients 1-<18 years), in which 45 patients were enrolled (30 plerixafor arm, 15 standard arm). Patient demographics and characteristics were well balanced across treatment arms. More patients in the plerixafor arm (24/30, 80%) met the primary endpoint of successful mobilization than in the standard arm (4/14, 28.6%, p = 0.0019). Adverse events reported as related to study treatment were mild, and no new safety concerns were identified. Plerixafor + standard G-CSF ± chemotherapy mobilization was generally well tolerated and efficacious when used to mobilize CD34+ cells in pediatric cancer patients.
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http://dx.doi.org/10.1038/s41409-020-0836-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452813PMC
September 2020

Refractory Epstein-Barr Virus (EBV)-Related Post-transplant Lymphoproliferative Disease: Cure by Combined Brentuximab Vedotin and Allogeneic EBV-Specific T-Lymphocytes.

Front Med (Lausanne) 2019 18;6:295. Epub 2019 Dec 18.

Department of Medicine, Hematology and Oncology, Ruhr-University Bochum, Bochum, Germany.

Post-transplant lymphoproliferative disease (PTLD) represents a serious complication following allogeneic hematopoietic stem cell transplantation (alloHSCT). Previously, survival rates of PTLD have improved due to the introduction of rituximab. However, reports on curative management of refractory PTLD are scarce. Today, there is no consensus how to treat rituximab-refractory PTLD, especially in highly aggressive disease. Here, we describe successful management of refractory EBV-associated PTLD, specifically DLBCL, with combined brentuximab vedotin and third-party EBV-specific T-cells in a multidisciplinary treatment approach.
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http://dx.doi.org/10.3389/fmed.2019.00295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930172PMC
December 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

Robust Identification of Suitable T-Cell Subsets for Personalized CMV-Specific T-Cell Immunotherapy Using CD45RA and CD62L Microbeads.

Int J Mol Sci 2019 Mar 20;20(6). Epub 2019 Mar 20.

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

Viral infections and reactivations remain a serious obstacle to successful hematopoietic stem cell transplantation (HSCT). When antiviral drug treatment fails, adoptive virus-specific T-cell transfer provides an effective alternative. Assuming that naive T cells (T) are mainly responsible for GvHD, methods were developed to generate naive T-cell-depleted products while preserving immune memory against viral infections. We compared two major strategies to deplete potentially alloreactive T cells: CD45RA and CD62L depletion and analyzed phenotype and functionality of the resulting CD45RA/CD62L naive T-cell-depleted as well as CD45RA⁺/CD62L⁺ naive T-cell-enriched fractions in the CMV pp65 and IE1 antigen model. CD45RA depletion resulted in loss of terminally differentiated effector memory T cells re-expressing CD45RA (T), and CD62L depletion in loss of central memory T cells (T). Based on these differences in target cell-dependent and target cell-independent assays, antigen-specific T-cell responses in CD62L-depleted fraction were consistently 3⁻5 fold higher than those in CD45RA-depleted fraction. Interestingly, we also observed high donor variability in the CD45RA-depleted fraction, resulting in a substantial loss of immune memory. Accordingly, we identified donors with expected response (DER) and unexpected response (DUR). Taken together, our results showed that a naive T-cell depletion method should be chosen individually, based on the immunophenotypic composition of the T-cell populations present.
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http://dx.doi.org/10.3390/ijms20061415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471767PMC
March 2019

Equally Interchangeable? How Sex and Gender Affect Transplantation.

Transplantation 2019 06;103(6):1094-1110

Liver Transplantation & Hepatology Unit, Hospital Universitario La Fe, IIS La Fe, University of Valencia-CIBEReHD, Valencia, Spain.

Organ transplantation as an option to overcome end-stage diseases is common in countries with advanced healthcare systems and is increasingly provided in emerging and developing countries. A review of the literature points to sex- and gender-based inequity in the field with differences reported at each step of the transplant process, including access to a transplantation waiting list, access to transplantation once waitlisted, as well as outcome after transplantation. In this review, we summarize the data regarding sex- and gender-based disparity in adult and pediatric kidney, liver, lung, heart, and hematopoietic stem cell transplantation and argue that there are not only biological but also psychological and socioeconomic issues that contribute to disparity in the outcome, as well as an inequitable access to transplantation for women and girls. Because the demand for organs has always exceeded the supply, the transplant community has long recognized the need to ensure equity and efficiency of the organ allocation system. In the spirit of equity and equality, the authors call for recognition of these inequities and the development of policies that have the potential to ensure that girls and women have equitable access to transplantation.
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http://dx.doi.org/10.1097/TP.0000000000002655DOI Listing
June 2019

Inhibition of Heme Oxygenase-1 Activity Enhances Wilms Tumor-1-Specific T-Cell Responses in Cancer Immunotherapy.

Int J Mol Sci 2019 Jan 23;20(3). Epub 2019 Jan 23.

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

Wilms tumor protein-1 (WT1) is an attractive target for adoptive T-cell therapy due to its expression in solid tumors and hematologic malignancies. However, T cells recognizing WT1 occur in low frequencies in the peripheral blood of healthy donors, limiting potential therapeutic possibilities. Tin mesoporphyrin (SnMP) is known to inhibit heme oxygenase-1 (HO-1), which has been shown to boost the activation and proliferation of human virus-specific T cells. We analyzed the influence of this effect on the generation of WT1-specific T cells and developed strategies for generating quantities of these cells from healthy donors, sufficient for adoptive T-cell therapies. HO-1 inhibition with SnMP increased WT1-specific T-cell frequencies in 13 (26%) of 50 healthy donors. To assess clinical applicability, we measured the enrichment efficiency of SnMP-treated WT1-specific T cells in response to a WT1-specific peptide pool and a HLA-A*02:01-restricted WT1 peptide by cytokine secretion assay. SnMP treatment resulted in a 28-fold higher enrichment efficacy with equal functionality. In conclusion, pharmacological inhibition of HO-1 activity with SnMP results in more efficient generation of functionally active WT1-specific T cells. This study demonstrates the therapeutic potentials of inhibiting HO-1 with SnMP to enhance antigen-specific T-cell responses in the treatment of cancer patients with WT1-positive disease.
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http://dx.doi.org/10.3390/ijms20030482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387130PMC
January 2019

Selective Effects of mTOR Inhibitor Sirolimus on Naïve and CMV-Specific T Cells Extending Its Applicable Range Beyond Immunosuppression.

Front Immunol 2018 17;9:2953. Epub 2018 Dec 17.

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

Cytomegalovirus (CMV) infection/reactivation remains among the most important complications of immunosuppression after transplantation. However, recent clinical observations indicate that mammalian target of rapamycin (mTOR) inhibition with sirolimus may improve the outcome of CMV complications. Underlying mechanisms of this observation, particularly the effect of sirolimus on naïve- and CMV-specific cytotoxic CD8 T-cell (CMV-CTL) functionality is still undiscovered. Here, the influence of sirolimus on naïve and memory CMV-CTLs was determined by CD3/CD28 crosslinking and alloreactivity assays. After stimulating CMV-CTL with HLA-A02:01-restricted CMVpp65-peptide loaded artificial antigen-presenting cells (aAPCs), we measured the effect of sirolimus on T-cell proliferation, phenotype, and functionality. Sirolimus significantly improved CMV-specific effector memory T-cell function and negatively influenced naïve T cells. This unique mechanism of action was further characterized by increased secretion of interferon-gamma (IFN-γ), granzyme B (GzB) and enhanced target-cell-dependent cytotoxic capacity of activated CMV-CTLs. Next-generation-sequencing (NGS) was applied to monitor T-cell receptor (TCR)-repertoire dynamics and to verify, that the increased functionality was not related to sirolimus-resistant CTL-clones. Instead, modulation of environmental cues during CMV-CTL development via IL-2 receptor (IL-2R)-driven signal transducer and activator of transcription-5 (STAT-5) signaling under mTOR inhibition allowed fine-tuning of T-cell programming for enhanced antiviral response with stable TCR-repertoire dynamics. We show for the first time that sirolimus acts selectively on human naïve and memory T cells and improves CMV-specific T-cell function via modulation of the environmental milieu. The data emphasize the importance to extend immune monitoring including cytokine levels and T-cell functionality which will help to identify patients who may benefit from individually tailored immunosuppression.
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http://dx.doi.org/10.3389/fimmu.2018.02953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304429PMC
October 2019

Clearance of Treatment Refractory Adenoviremia via Adenovirus-specific Donor T-Cell Transfer During Aplasia After αβTCR-CD19-Depleted Stem Cell Transplantation.

Clin Infect Dis 2019 04;68(8):1406-1409

Division of Pediatric Stem Cell Therapy, Clinic for Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine University, Medical Faculty, Düsseldorf.

Here, we report the case of severe adenoviremia in a 7-year-old boy with highly-resistant, acute leukemia. A combined approach of αβTCR-CD19-depleted stem cell transplantation, enabling immunosuppression-free post-transplant care, and early transfer of adenovirus-specific donor T cells during aplasia resulted in rapid and complete clearance of the treatment-refractory adenoviremia.
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http://dx.doi.org/10.1093/cid/ciy820DOI Listing
April 2019

Mini photopheresis for refractory chronic graft-versus-host disease in children and adolescents.

Transfusion 2018 11 6;58(11):2495-2500. Epub 2018 Oct 6.

Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University, Giessen, Germany.

Background: Extracorporeal photopheresis (ECP) has been established to treat graft-versus-host disease. Our mini ECP technique (mini-ECP) allows for treatment of patients with GVHD and contraindications for classical ECP or low body weight. The safety and efficacy of applying ECP for the long-term treatment of chronic GVHD (cGVHD) have not been described.

Study Design And Methods: A retrospective analysis of mini-ECP treatments for children and adolescents with cGVHD was performed. Mini-ECP with 100 to 200 mL of whole blood was used to treat 14 patients. The median age at the start of treatment was 7 years (range, 1-17 years), and median body weight was 20 kg (range, 8-53 kg). A total of 703 mini-ECP treatments was performed. The median number of treatments per patient was 35 (range, 8-129), and median treatment duration was 11 months (range, 1.4-28.5 months).

Results: Mini-ECP was well tolerated. Four adverse events occurred in three patients, and two of them were related to the ECP procedure. Complete or partial responses were observed in 10 patients. Steroids were discontinued in seven patients and tapered in three others. Responses were seen in the skin, mouth, gastrointestinal tract, and eyes.

Conclusion: Mini-ECP represents a less invasive ECP alternative for low-body-weight patients with cGVHD and contraindications for apheresis.
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http://dx.doi.org/10.1111/trf.14880DOI Listing
November 2018

Dissecting Epstein-Barr Virus-Specific T-Cell Responses After Allogeneic EBV-Specific T-Cell Transfer for Central Nervous System Posttransplant Lymphoproliferative Disease.

Front Immunol 2018 27;9:1475. Epub 2018 Jun 27.

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

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) with central nervous system (CNS) involvement is a severe complication after solid organ transplantation. Standard treatment with reduction of immunosuppression and anti-CD20 antibody application often fails leading to poor outcome. Here, we report the case of an 11-year-old boy with multilocular EBV-positive CNS PTLD 10 years after liver transplantation. Complete remission was achieved by repeated intravenous and intrathecal anti-CD20 antibody rituximab administration combined with intrathecal chemotherapy (methotrexate, cytarabine, prednisone) over a time period of 3 months. Due to the poor prognosis of CNS PTLD and lack of EBV-specific T-cells (EBV-CTLs) in patient's blood, we decided to perform EBV-directed T-cell immunotherapy as a consolidating treatment. The patient received five infusions of allogeneic EBV-CTLs from a 5/10 HLA-matched unrelated third-party donor. No relevant acute toxicity was observed. EBV-CTLs became detectable after first injection and increased during the treatment course. Next-generation sequencing (NGS) TCR-profiling verified the persistence and expansion of donor-derived EBV-specific clones. After two transfers, epitope spreading to unrelated EBV antigens occurred suggesting onset of endogenous T-cell production, which was supported by detection of recipient-derived clones in NGS TCR-profiling. Continuous complete remission was confirmed 27 months after initial diagnosis.
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http://dx.doi.org/10.3389/fimmu.2018.01475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030255PMC
June 2018

Personalized adoptive immunotherapy for patients with EBV-associated tumors and complications: Evaluation of novel naturally processed and presented EBV-derived T-cell epitopes.

Oncotarget 2018 Jan 21;9(4):4737-4757. Epub 2017 Dec 21.

Institute for Transfusion Medicine, Hannover Medical School (MHH), Hanover, Germany.

Morbidity and mortality of immunocompromised patients are increased by primary infection with or reactivation of Epstein-Barr virus (EBV), possibly triggering EBV post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) promises a non-toxic immunotherapy to effectively prevent or treat these complications. To improve immunotherapy and immunomonitoring this study aimed at identifying and evaluating naturally processed and presented HLA-A*03:01-restricted EBV-CTL epitopes as immunodominant targets. More than 15000 peptides were sequenced from EBV-immortalized B cells transduced with soluble HLA-A*03:01, sorted using different epitope prediction tools and eleven candidates were preselected. T2 and Flex-T peptide-binding and dissociation assays confirmed the stability of peptide-MHC complexes. Their immunogenicity and clinical relevance were evaluated by assessing the frequencies and functionality of EBV-CTLs in healthy donors ( > 10) and EBV PTLD-patients ( = 5) by multimer staining, Eli- and FluoroSpot assays. All eleven peptides elicited EBV-CTL responses in the donors. Their clinical applicability was determined by small-scale T-cell enrichment using Cytokine Secretion Assay and immunophenotyping. Mixtures of these peptides when added to the EBV Consensus pool revealed enhanced stimulation and enrichment efficacy. These EBV-specific epitopes broadening the repertoire of known targets will improve manufacturing of clinically applicable EBV-CTLs and monitoring of EBV-specific T-cell responses in patients.
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http://dx.doi.org/10.18632/oncotarget.23531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797009PMC
January 2018

Cardiovascular risk factors and subclinical organ damage after hematopoietic stem cell transplantation in pediatric age.

Bone Marrow Transplant 2018 08 9;53(8):983-992. Epub 2018 Feb 9.

Department of Paediatric Hematooncology, Clinic for Paediatrics III, University Hospital Duisburg-Essen, Essen, Germany.

Advances in allogeneic hematopoietic stem cell transplantation (HSCT) in malignant and non-malignant diseases result in more long-term survivors, in whom cardiovascular (CV) disease is one leading non-cancer cause of death. This study aimed to evaluate risk factors and subclinical CV organ damage in survivors after HSCT in pediatric age. We enrolled 64 children in a cross-sectional approach 3.3 ± 3.1 years after HSCT. Anthropometric data, laboratory values, office and 24-h ambulatory blood pressure monitoring (ABPM) were evaluated, showing a high prevalence of obesity, hypertension and dyslipidemia. CV organ damage was determined by non-invasive measurements of aortic pulse wave velocity (PWV), left ventricular mass index (LVMI), and carotid intima media thickness (IMT). Increased IMT and elevated PWV reflecting subclinical vascular damage were detected in 48% (IMT) and 6% (PWV) of our population. For IMT, physical activity had a positive impact and was worsened by time after HSCT. Our results show a surprisingly high rate of subclinical CV organ damage and classical risk factors. Therefore, diagnosis and management of well-known CV risk factors belong to clinical care after HSCT.
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http://dx.doi.org/10.1038/s41409-018-0104-xDOI Listing
August 2018

Graft outcomes following diagnosis of post-transplant lymphoproliferative disease in pediatric kidney recipients: a retrospective study.

Transpl Int 2018 04 11;31(4):367-376. Epub 2017 Oct 11.

Department of Pediatric Nephrology, Hannover Medical School, Hannover, Germany.

Data related to graft outcomes following post-transplant lymphoproliferative disease (PTLD) in pediatric kidney transplantation are scarce. Data were analyzed retrospectively from 12 children (eight boys) for 3 years after diagnosis of PTLD, with a loss of follow-up after 2 years in two of 12. In all cases, intensity of immunosuppressive therapy was reduced, which switched from calcineurin inhibitor to a mammalian target of rapamycin (mTOR) inhibitor in ten cases. Nine children were treated with six doses of rituximab according to the PED-PTLD-2005 protocol, with additional treatment in one child as per protocol. One patient received EuroNet-PHL C1. In four patients, donor-specific antibodies were detected after PTLD diagnosis at 3, 4, 5 and 7 years, respectively. One patient developed chronic antibody-mediated rejection (cAMR) 12 years after diagnosis, losing the graft 1 year later. Three patients with recurrence of the original disease also lost their grafts, one at the time of diagnosis of PTLD, and two after 4 years. Range-based analysis of variance showed that there was no decrease in estimated GFR at 1, 2, or 3 years after diagnosis of PTLD (P = 0.978). In conclusion, treatment of PTLD with reduced immunosuppression is safe and efficient. This may be due to B-cell-depleting therapy of PTLD with rituximab.
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http://dx.doi.org/10.1111/tri.13071DOI Listing
April 2018

Variant of classical high grade PTLD: post-transplant EBV-negative T cell lymphoblastic leukaemia after solid organ transplantation.

Ann Hematol 2017 Aug 14;96(8):1403-1405. Epub 2017 Jun 14.

Institute of Pathology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.

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http://dx.doi.org/10.1007/s00277-017-3026-6DOI Listing
August 2017

Early and Late Factors Impacting Patient and Graft Outcome in Pediatric Liver Transplantation: Summary of an ESPGHAN Monothematic Conference.

J Pediatr Gastroenterol Nutr 2017 09;65(3):e53-e59

*Swiss Center for Liver Disease in Children, University Hospitals Geneva, Geneva, Switzerland †Division of Infectious Diseases and the Transplant and Regenerative Medicine Centre, Hospital for Sick Children, University of Toronto, Toronto, Canada ‡Pediatric Nephrology, Imagine Institute, Necker-Enfants Malades Hospital, APHP, Paris Descartes University-Sorbonne Paris Cité, Paris, France §Pediatric Liver Care Center, Cincinnati Children's Hospital, Cincinnati, OH ||Department of Surgery, Ospedale Papa Giovanni XXIII, Bergamo, Italy ¶Faculté de Médecine, University de Nantes, Nantes, France #Pediatric Hepatology, Gastroenterology and Transplantation, Ospedale Papa Giovanni XXIII, Bergamo, Italy **Pediatric Hepatology Unit, Necker Enfants Malades Hospital, Paris, France ††First Department of Pediatrics, Semmelweis University, Budapest, Hungary ‡‡University Tor Vergata, Rome, Italy §§Pediatric Liver GI and Nutrition Centre, King's College Hospital, London, UK ||||Faculty of Medicine, Istanbul University, Istanbul, Turkey ¶¶Institute of Transplant Immunology, FIB-Tx, Hannover Medical School, Germany ##Abdominal Transplant Surgery, University of California San Francisco, San Francisco, CA ***Department of Pediatrics, Karolinska University Hospital, CLINTEC Karolinska Institutet, Stockholm, Sweden †††Pediatric Radiology Department, Hôpital Bicêtre, Hôpitaux Universitaires Paris Sud- Assistance Publique Hôpitaux de Paris, Le Kremlin-Bicêtre, France ‡‡‡Pediatric Hepatology and Liver Transplantation, Hospital Universitario Infantil La Paz, Madrid, Spain §§§Department of Pediatrics, University Children's Hospital Bonn, Bonn, Germany ||||||Center for Cell and Gene Therapy and Texas Children's Cancer Center, Texas Children's Hospital, Houston Methodist Hospital, Baylor College of Medicine, Houston, TX ¶¶¶Pediatric Liver GI and Nutrition Centre, King's College, London, UK ###Seattle Children's Hospital and University of Washington, Seattle, WA ****School of Cancer Sciences ††††Department of Cellular Pathology, Queen Elizabeth Hospital, University of Birmingham, Birmingham, UK ‡‡‡‡The European Transplant Registry, APHP Paul Brousse Hospital, Villejuif, France §§§§Liver Unit, Birmingham Children's Hospital, Birmingham, UK ||||||||Department of Pediatric Hematology/Oncology and Integrated Research and Treatment Center for Transplantation, Hannover Medical School, Germany ¶¶¶¶Hillman Center for Pediatric Transplantation, Children's Hospital of Pittsburgh, Pittsburgh, PA ####Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", Section of Pediatrics, University of Salerno, Baronissi, Italy *****Bambino Gesu Children's Hospital, Rome, Italy †††††Department of Pediatric Gastroenterology, Hepatology, and Nutrition, Ege University, Izmir, Turkey ‡‡‡‡‡Pediatric Surgery and Transplantation Unit, Cliniques Universitaires de Saint-Luc, Université Catholique de Louvain, Brussels, Belgium §§§§§ISMETT, University of Pittsburgh Medical Center, Palermo, Italy ||||||||||The Children's Memorial Health Institute, Warsaw, Poland ¶¶¶¶¶Divisions of Abdominal and Transplant Surgery, Faculty of Medicine and University Hospitals Geneva, Geneva, Switzerland #####University of Salerno, Baronissi, Italy ******King's College Hospital, Institute of Liver Studies, London, UK ††††††Department for Pediatric Kidney, Liver and Metabolic Disease, Division of Pediatric Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany.

As pediatric liver transplantation comes of age, experts gathered to discuss current paradigms and define gaps in knowledge warranting research to further improve patient and graft outcomes. Identified areas ripe for collaborative research include understanding the molecular and cellular mechanisms of tolerance and the role of donor-specific antibodies, considering ways to expand donor pool, minimizing long-term side effects of immunosuppression, and fine-tuning surgical techniques to minimize biliary and vascular complications.
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http://dx.doi.org/10.1097/MPG.0000000000001564DOI Listing
September 2017

Comparative Analysis of Clinical-Scale IFN-γ-Positive T-Cell Enrichment Using Partially and Fully Integrated Platforms.

Front Immunol 2016 30;7:393. Epub 2016 Sep 30.

Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.

Background And Aims: The infusion of enriched CMV-specific donor T-cells appears to be a suitable alternative for the treatment of drug-resistant CMV reactivation or infection after both solid organ and hematopoietic stem cell transplantation. Antiviral lymphocytes can be selected from apheresis products using the CliniMACS Cytokine-Capture-System either with the well-established CliniMACS Plus (Plus) device or with its more versatile successor CliniMACS Prodigy (Prodigy).

Methods: Manufacturing of CMV-specific T-cells was carried out with the Prodigy and Plus in parallel starting with 0.8-1 × 10 leukocytes collected by lymphapheresis ( = 3) and using the MACS GMP PepTivator HCMVpp65 for antigenic restimulation. Target and non-target cells were quantified by a newly developed single-platform assessment and gating strategy using positive (CD3/CD4/CD8/CD45/IFN-γ), negative (CD14/CD19/CD56), and dead cell (7-AAD) discriminators.

Results: Both devices produced largely similar results for target cell viabilities: 37.2-52.2% (Prodigy) vs. 51.1-62.1% (Plus) CD45/7-AAD cells. Absolute numbers of isolated target cells were 0.1-3.8 × 10 viable IFN-γ CD3 T-cells. The corresponding proportions of IFN-γ CD3 T-cells ranged between 19.2 and 95.1% among total CD3 T-cells and represented recoveries of 41.9-87.6%. Within two parallel processes, predominantly IFN-γ CD3CD8 cytotoxic T-cells were enriched compared to one process that yielded a higher amount of IFN-γ CD3CD4 helper T lymphocytes. T-cell purity was higher for the Prodigies products that displayed a lower content of contaminating IFN-γ T-cells (3.6-20.8%) compared to the Plus products (19.9-80.0%).

Conclusion: The manufacturing process on the Prodigy saved both process and hands-on time due to its higher process integration and ability for unattended operation. Although the usage of both instruments yielded comparable results, the lower content of residual IFN-γ T-cells in the target fractions produced with the Prodigy may allow for a higher dosage of CMV-specific donor T-cells without increasing the risk for graft-versus-host disease.
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http://dx.doi.org/10.3389/fimmu.2016.00393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5044705PMC
September 2016

Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection.

J Transl Med 2016 10 7;14(1):286. Epub 2016 Oct 7.

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

Background: Human adenovirus (HAdV) infections remain a significant cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Efficient antiviral T-cell responses are necessary to clear infection, which is hampered by delayed immune reconstitution and medical immunosuppression after HSCT. Protective immunity may be conferred by adoptive transfer of HAdV-specific T cells. For identification of patients at risk and monitoring of treatment responses diligent assessment of anti-HAdV cellular immune responses is crucial. The HAdV-derived protein hexon has been recognized as a major immunodominant target across HAdV species. We aimed at identifying further targets of protective anti-HAdV immune response and characterizing immunogenic epitopes.

Methods: Nineteen candidate nonamers from hexon and penton proteins were identified by epitope binding prediction. Peptides were synthesized and tested for in vivo immunogenicity by screening peripheral blood mononuclear cells from healthy volunteers (n = 64) and HAdV-infected stem cell recipients (n = 26) for memory T cells recognizing the candidate epitopes in the context of most common HLA alleles.

Results: Functional CD8 T cells recognizing seven epitopes were identified, among them four penton-derived and two hexon-derived peptides. The HLA-A*01-restricted penton-derived peptide STDVASLNY (A01Penton) and HLA-A*02-restricted hexon-derived peptide TLLYVLFEV (A02Hexon) were recognized by more than half of the persons carrying the respective HLA-type.

Conclusions: Thus, the HAdV-derived penton protein is a novel major target of the anti-HAdV immune response. Identification of new immunodominant epitopes will facilitate and broaden immune assessment strategies to identify patients suitable for T-cell transfer. Knowledge of additional target structures may increase T-cell recovery in manufacturing processes.
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http://dx.doi.org/10.1186/s12967-016-1042-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055684PMC
October 2016

Classical Hodgkin lymphoma-type PTLD after solid organ transplantation in children: a report on 17 patients treated according to subsequent GPOH-HD treatment schedules.

Leuk Lymphoma 2017 03 11;58(3):633-638. Epub 2016 Aug 11.

a Department of Pediatric Hematology and Oncology , Hannover Medical School , Hannover , Germany.

Post-transplant lymphoproliferative disease (PTLD) is a severe complication after solid organ transplantation (SOT). Classical Hodgkin lymphoma-type (HL-) PTLD is a rare subtype, and systematic data on treatment and prognosis are lacking. We report on 17 pediatric patients with classical HL-PTLD. HL-PTLD developed late at a median of 8.1 years after SOT. It was commonly EBV-positive (16/17) and expressed both CD30 (all tumors) and CD20 (8/17 tumors). Patients were treated with chemotherapy +/- involved field radiotherapy (IF-RT) according to the respective GPOH-HD protocol tailored by stage and LDH. Overall survival at 2 and 5 years was 86% with 81% of patients surviving event-free. Six patients had additional rituximab treatment; in two it was given as upfront monotherapy and in four was given concurrently with their chemotherapy. Rituximab monotherapy did not lead to long-term remission. In conclusion, treatment of HL-PTLD with classical HL chemotherapy is effective and tolerable. New treatment modalities such as CD30-targeted or EBV-specific agents may diminish toxicity.
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http://dx.doi.org/10.1080/10428194.2016.1205742DOI Listing
March 2017

Post-transplant lymphoproliferative disorders with naso- and oropharyngeal manifestation.

Transpl Int 2015 Nov 30;28(11):1299-307. Epub 2015 Jul 30.

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

The nasopharyngeal/oropharyngeal lymphatic tissues represent the anatomical site of Epstein-Barr virus (EBV) entry. Post-transplant lymphoproliferative disorders (PTLD) are often associated with EBV, but little is known about the characteristics of nasopharyngeal/oropharyngeal mass-forming PTLD. Retrospective evaluation of our own PTLD database (n = 79) and the PubMed(®) database (n = 61) has been performed. Sinonasal/oro-/nasopharyngeal lymphatic masses were early lesions (n = 54/140, 38.5%), polymorphic PTLD (n = 32/140, 23%), monomorphic B-PTLD (n = 47/140, 33.5%) and T-PTLD (n = 7/140, 5%). One-fourth of lesions manifested as masses in the Waldeyer's ring, and in two-thirds of cases, swelling of tonsils was related to manifestation of benign early lesions. Tonsil infiltration by polymorphic PTLD and monomorphic PTLD was present in one-third of cases. Extratonsillar masses were mainly monomorphic PTLD. Meta-analysis of our data in combination with previously published data revealed that lung transplantation and young patients are at a higher risk for earlier manifestation of monomorphic PTLD. Therapy is similar to PTLD therapy strategies, in general reduced immunosuppression and chemotherapy for polymorphic and monomorphic PTLD, and diagnostic and therapeutic surgical gross tumour resection of tonsillar/adenoid lesions. In summary, it is relevant for the clinical differential diagnosis that oro-/nasopharyngeal aggressive PTLD manifested in ~30% as tonsillar masses and >90% at extratonsillar sites.
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http://dx.doi.org/10.1111/tri.12632DOI Listing
November 2015

Broad spectrum antiviral T cells for viral complications after hematopoietic stem cell transplantation.

Ann Transl Med 2015 May;3(Suppl 1):S4

1 Department of Pediatric Hematology and Oncology, 2 Integrated Research and Treatment Center Transplantation (IFB-Tx), 3 Institute for Transfusion Medicine, Hannover Medical School, 30625 Hannover, Germany.

Major complications of hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT), such as graft rejection and graft-versus-host-disease (GvHD), are countered by suppressing the host immune system via chemotherapy and radiation, immunosuppressive drugs, or conditioning regimens such as in vivo or in vitro T-cell depletion. While immunocompromised, the patient is rendered susceptible to a number of viral infections and reactivations mainly caused by endogenous herpes viruses like cytomegalovirus (CMV) and Epstein-Barr virus (EBV) and by lytic agents such as adenovirus (ADV). In the paper entitled "Activity of broad-spectrum T cells as treatment for ADV, EBV, CMV, BKV, and HHV6 Infections after HSCT" published recently in Science Translational Medicine, Anastasia Papadopoulou and colleagues reported a suitable technology for rapid generation of antiviral T cells with a broad specificity in a single-culture for clinical application. In a small clinical trial with 11 patients they demonstrated safety and efficacy of adoptive multivirus-specific T-cell transfer.
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http://dx.doi.org/10.3978/j.issn.2305-5839.2015.01.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437961PMC
May 2015

Adoptive T-cell therapy with hexon-specific Th1 cells as a treatment of refractory adenovirus infection after HSCT.

Blood 2015 Mar 23;125(12):1986-94. Epub 2015 Jan 23.

Department of Pediatric Hematology/Oncology, University Children's Hospital Tübingen, Tübingen, Germany; German Center for Infection Research, Ludwig Maximilians University Munich, Munich, Germany.

Hematopoietic stem cell transplantation (HSCT) has improved over the last few decades. However, viral infections are often refractory to pharmacologic treatment and require alternative treatment strategies such as immunotherapy. Adenovirus (AdV) is th predominant disease-causing pathogen in pediatric HSCT. In a clinical trial, we analyzed safety and efficacy of ex vivo adoptive T-cell transfer (ACT) with hexon-specific T cells, predominantly of the T-helper cell 1 (Th1) phenotype, in 30 patients with AdV disease or viremia. ACT was feasible with no acute toxicities or significant onset of graft-versus-host disease. ACT led to in vivo antiviral immunity for up to 6 months with viral control, resulting in complete clearance of viremia in 86% of patients with antigen-specific T-cell responses. After ACT and a follow-up of 6 months, overall survival was markedly increased in responders (mean, 122 days; 15 survivors) compared with nonresponders who all died shortly after ACT (mean, 24 days; no survivors). AdV-related mortality was 100% in nonresponders compared with 9.5% in responders (≥1 log reduction of DNA copies per milliliter after ACT). In summary, ex vivo ACT of AdV-specific Th1 cells was well tolerated and led to successful and sustained restoration of T-cell immunity correlated with virologic response and protection from virus-related mortality. This cellular immunotherapy is a short-term available and broadly applicable treatment. The study is registered at European Union Clinical Trials Register as 2005-001092-35.
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http://dx.doi.org/10.1182/blood-2014-06-573725DOI Listing
March 2015

Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells.

J Transl Med 2014 Dec 16;12:336. Epub 2014 Dec 16.

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

Background: The adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6.

Methods: To obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ(+) T cells exceeded 0.03% of CD3(+) lymphocytes as determined by IFN-γ cytokine secretion assay.

Results: Starting with low concentration of IFN-γ(+) T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ(+)CD3(+) T cells (1.42 × 10(6), 0.05 × 10(6), and 1.15 × 10(6)) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8(+) than CD4(+) memory T cells, both of which were effectively enriched to a total of 81.0% CD8(+)IFN-γ(+) and 38.4% CD4(+)IFN-γ(+) T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes.

Conclusions: The generation of antiviral CD4(+) and CD8(+) T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.
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http://dx.doi.org/10.1186/s12967-014-0336-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335407PMC
December 2014

CD30 in pediatric post-transplant lymphoproliferative disease after solid organ transplant: characterization of a new therapeutic target.

Leuk Lymphoma 2015 Mar 3;56(3):832-3. Epub 2014 Nov 3.

Dr. von Hauner Children's Hospital, Department of Pediatrics, Ludwig-Maximilians-University , Munich , Germany.

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http://dx.doi.org/10.3109/10428194.2014.941837DOI Listing
March 2015

Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein-Barr virus-specific T cells as targets of interest in immunotherapeutic approaches.

J Immunol Methods 2014 Jun 28;408:101-13. Epub 2014 May 28.

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany; Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany. Electronic address:

Adoptive immunotherapy with donor-derived antiviral T cells can prevent viral complications such as with cytomegalovirus (CMV) and Epstein-Barr virus (EBV). In this context accurate monitoring of cellular immunity is essential and requires suitable quantitative and qualitative assays for high-throughput screening. We comparatively analyzed 57 HLA-typed healthy donors for memory T-cell responses to CMV- and EBV-derived proteins, peptide pools and single HLA-restricted peptides by five commonly used immunoassays in parallel: enzyme-linked immunospot (ELISPOT), cytokine secretion assay (CSA), intracellular cytokine staining (ICS), enzyme-linked immunosorbent assay (ELISA) and pMHC multimer staining. T-cell responses varied greatly between the different target antigens in the investigated assays. IFN-γ ELISPOT consistently detected the highest T-cell response levels against CMV and EBV. CMV-specific T cells were detected in 100% of CMV-seropositive donors tested using CMVpp65 protein and/or overlapping CMVpp65 peptide pool. CMV-specific T cells in HLA-A*02:01-positive/CMV-seropositive donors were identified directly by HLA-A02/CMVpp65 (A02pp65) multimer staining and, after short in vitro stimulation with HLA-A*02:01-restricted pp65 peptide, by ELISPOT, ELISA, ICS and CSA. A peptide-specific T-cell response was detected in only 4 HLA-A*02:01-positive donors (50%). Despite A02pp65 peptide negativity, T-cell responses to CMVpp65 protein and/or overlapping peptide pool were detected. Comparing the specific immune response against EBV antigens in healthy donors overall, BZLF1-specific T cells (<92.9% peptides, <56.3% peptide pool) were more frequent than EBNA-specific T cells (<64.3% peptides, <46.9% peptide pool) with higher percentage of positive findings for single HLA-restricted EBV peptides. T-cell response against HLA-B*08 peptide epitopes was predominant (multimer staining: EBNA3A: 9/14 and BZLF1: 7/14, IFN-γ ELISPOT: EBNA3A: 13/14 and BZLF1: 11/14). The fact that responses to EBV-specific antigens were not detected in every single EBV-seropositive donor as well as that the T-cell frequencies in response to the investigated EBV antigens differed strongly in the donor cohort indicates that these epitopes are less immunodominant than CMVpp65. Taken together, precise monitoring of T-cell immunity against infectious agents in potential T-cell donors and post-transplant recipients requires individual selection of antigens and immunoassays for the efficient detection and generation of clinically relevant T cells. Due to its lower detection limit and direct visualization of each IFN-γ-secreting cell we identified ELISPOT analysis to be preferable for high-throughput pre-screening. CSA was found to be advantageous for a more detailed analysis of antigen-specific T-cell subsets.
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http://dx.doi.org/10.1016/j.jim.2014.05.011DOI Listing
June 2014