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The objective of this study was to establish a mobilization and apheresis regimen for collection of two consecutive polymorphonuclear neutrophil (PMN) concentrates from the same donor. In this prospective study, 111 healthy unrelated volunteers underwent either one (Group 1, n = 57) or two consecutive granulocyte apheresis procedure (Group 2, n = 54) using the a cell separator (Spectra). Both Group 1 and 2 donors were initially mobilized with glycosylated G-CSF 6.0 µg per kg (range, 5.2-7.0 µg/kg) subcutaneously plus oral dexa-methasone (DXM, 8 mg) and underwent granulocyte apheresis (GA-1) 16 hours (range, 13-18 hr) after initial G-CSF+DXM. Group 2 donors were remobilized with a second DXM dose of 8 mg (n = 13), 4 mg (n = 15), 1.5 mg (n = 13), or none (n = 13), and a second apheresis (GA-2) was run 40 hours (range, 37-42 hr) after G-CSF+DXM administration and 12 hours after remobilization with DXM alone. Based on equivalent median preapheresis WBC and PMN counts of around 35 × 109 WBCs per L and 33 × 109 PMNs per L after initial mobilization the GA-1 yields were 85 × 109 PMNs per U (range, 34-150) in Group 1 and 75 × 109 PMNs per U (range, 35-135) in Group 2 (p = 0.14, NS). In Group 2, median preapheresis values of 19.6 × 109 WBCs per L (range, 9.5-37.0) and 16.6 × 109 PMNs per L (range, 8.8-34.8) were measured after remobilization and GA-2 yields of 49 × 109 WBCs per U (range, 26-113) and 42 × 109 PMNs per U (range, 21-95) were obtained. Borderline statistical differences in the GA-2 yields were observed from the remobilized donors: 8 mg: 36 × 109 PMNs per U (range, 23-60); 4 mg: 47 × 109 PMNs per U (range, 21-56) (p ≤ 0.04); 1.5 mg: 45 × 109 PMNs per U (range, 23-79); placebo: 36 × 109 PMNs per U (range, 26-95) (p ≤ 0.04). Two consecutive neutrophil concentrates for transfusion can be obtained after single stimulation with Lenograstim+DXM. A second application of DXM alone is necessary, but of limited value. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to find a regimen for mobilization and collection of granulocytes that combines low-dose G-CSF administration with satisfactory PMN mobilization and apheresis at a low rate of donor adverse reactions.Study Design and Methods: In a prospective study, 52 healthy unrelated volunteers received a single subcutaneous injection of glycosylated G-CSF (Lenograstim Chugai-Pharma, Frankfurt, Germany) at medians of 3.1 (range, 2.4-3.6) microg per kg plus dexamethasone (8 mg orally; n = 29) or at 11.8 (7.1-18.5) microg of lenograstim per kg (p < or = 0.0001) without dexamethasone (n = 23) and underwent standard apheresis using the PMN program of a cell separator (Spectra, COBE [now Gambro] BCT). WBC and PMN mobilization results and apheresis yields were compared and the severity and clinical significance of donor adverse reactions was evaluated.Results: For the low-dose G-CSF plus dexamethasone versus the high-dose G-CSF alone group, similar mobilization results were observed for WBCs with 31.3 (19.1-44.9) x 10(9) per L versus 27.5 (19.2-44.0) x 10(9) per L (p = 0.21, NS) and PMNs with 29.0 (17.6-42.2) x 10(9) per L versus 25.2 (16.2-39.0) x 10(9) per L (p = 0.08, NS). The PMN apheresis yields were equal with 70 (39-139) x 10(9) per unit with low-dose G-CSF versus 68 (33-120) x 10(9) per unit in the high-dose G-CSF group (p = 0.83, NS). Regarding donor adverse reactions, 7 out of 29 (24%) and 8 out of 23 donors (35%) reported moderate or severe symptoms. The character of these reactions was different; symptoms of greater clinical significance and a higher need for analgesics were observed in the high-dose G-CSF group.Conclusions: A Lenograstim dose of 3 microg per kg plus DXM assures effective PMN mobilization and acceptable apheresis components. The combination of glycosylated G-CSF with DXM allows a significant dose reduction in G-CSF for PMN mobilization and collection as compared with higher G-CSF doses alone. In the high-dose G-CSF mobilization group, adverse reactions were more severe and required more analgesics. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the impact of glycosylated granulocyte colony stimulating factor (G-CSF, Lenograstim) dosage with or without (w/o) dexamethasone (DXM) for mobilisation and collection of granulocytes for subsequent transfusion. Donors and methods: In a prospective randomized trial, 64 healthy unrelated blood donors received a single dose of G-CSF sc at a dose range of 3-14 mg/kg ± DXM. The donors underwent a standard granulocyte apheresis using the Spectra cell separator (separation factor 250, anticoagulation 6%-HES/33%-Na-Citrate, processed donor blood volume 7 L). Donor white blood cell (WBC) and granulocyte counts (GC), granulocyte apheresis yields (G-Y) were compared and donor complaints were evaluated by a questionnaire. Results in medians (ranges): 3mg/kg + DXM (14 donors): WBC 31.4 (19.2-46.9)'10*E3/mL, GC 29.6 (17.6-43.0)'10*E3/mL, G-Y 7.1 (3.9-10.0)'10*E10/unit; 6mg/kg + DXM (14 donors): WBC 35.9 (8.551.9)'10*E3/mL, GC 32.7 (2.3-47.3)'10*E3/mL, G-Y 9.3 (3.4-15.0)'10'E10/ unit; 14mg/kg + DXM (12 donors): WBC 35.1 (21.9-48.8)'10*E3/mL, 32.6 (19.744.4)'10*E3/mL, G-Y 8.3 (4.2-14.7)'10*E 10/unit; 9mg/kg w/o DXM (12 donors): WBC 26.9 (19.2-44.0)'10*E3/mL, 23.5 (16.5-39.1)'10*E3/mL, G-Y 6.3 (3.311.0)'10*E10/unit; 13 mg/kg w/o DXM (12 donors): WBC 29.7 (20.140.4)'10*E3/mL, GC 26.3 (17.0-35.0)'10*E3/mL, G-Y 7.8 (4.2-12.0)'10*E10/ unit. In the G-CSF+DXM group 22/40 donors (55%) had no complaints at all, 18/40 (45%) presented mild G-CSF related toxicity WHO grade I-II. In the donors without DXM 5 (21%) had no complaints, 16 (67%) had moderate GCSF related toxicity WHO grade I-III and 3 (13%)developed severe toxicity with heavy bone pain (n=2) and pain related to the spleen (n=1) so that they were unable to follow their professional duties for 1-3 days. Conclusions: Single Lenograstim doses of 6 mg/kg plus DXM offer the best dose/bone marrow response relationship for granulocyte mobilisation and assure apheresis products with high granulocyte yields. Regardless of DXM administration higher G-CSF doses up to 14 mg/kg ara of no advantage for GC mobilisation and collection, but can be associated with unacceptable toxicity for unrelated healthy donors. [ABSTRACT FROM AUTHOR]

Background: Immune cell dysfunction is a central part of immune paralysis in sepsis. Granulocyte concentrate (GC) transfusions can induce tissue damage via local effects of neutrophils. The hypothesis of an extracorporeal plasma treatment with granulocytes is to show beneficial effects with fewer side effects. Clinical trials with standard GC have supported this approach. This ex vivo study investigated the functional properties of purified granulocyte preparations during the extracorporeal plasma treatment., Methods: Purified GC were stored for up to 3 days and compared with standard GC in an immune cell perfusion therapy model. The therapy consists of a plasma separation device and an extracorporeal circuit. Plasma is perfused through the tubing system with donor immune cells of the GC, and only the treated plasma is filtered for re-transfusion. The donor immune cells are retained in the extracorporeal system and discarded after treatment. Efficacy of granulocytes regarding phagocytosis, oxidative burst as well as cell viability and metabolic parameters were assessed., Results: In pGC, the metabolic surrogate parameters of cell functionality showed comparable courses even after a storage period of 72 h. In particular, glucose and oxygen consumption were lower after extended storage. The course of lactate dehydrogenase concentration yields no indication of cell impairment in the extracorporeal circulation. The cells were viable throughout the entire study period and exhibited preserved phagocytosis and oxidative burst functionality., Conclusion: The granulocytes demonstrated full functionality in the 6 h extracorporeal circuits after 3 days storage and in septic shock plasma. This is demonstrating the functionality of the system and encourages further clinical studies., (© 2023 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

BACKGROUNDAdoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications.METHODSWe provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis.RESULTSForty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients' blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response.CONCLUSIONPersonalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction.TRIAL REGISTRATIONNot applicable.FUNDINGThis study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).

Objectives: Evaluation of the feasibility of SARS-CoV-2-specific T cell manufacturing for adoptive T cell transfer in COVID-19 patients at risk to develop severe disease. Methods: Antiviral SARS-CoV-2-specific T cells were detected in blood of convalescent COVID-19 patients following stimulation with PepTivator SARS-CoV-2 Select using Interferon-gamma Enzyme-Linked Immunospot (IFN-γ ELISpot), SARS-CoV-2 T Cell Analysis Kit (Whole Blood) and Cytokine Secretion Assay (CSA) and were characterized with respect to memory phenotype, activation state and cytotoxic potential by multicolor flow cytometry, quantitative real-time PCR and multiplex analyses. Clinical-grade SARS-CoV-2-specific T cell products were generated by stimulation with MACS GMP PepTivator SARS-CoV-2 Select using CliniMACS Prodigy and CliniMACS Cytokine Capture System (IFN-gamma) (CCS). Functionality of enriched T cells was investigated in cytotoxicity assays and by multiplex analysis of secreted cytotoxic molecules upon target recognition. Results: Donor screening via IFN-γ ELISpot allows for pre-selection of potential donors for generation of SARS-CoV-2-specific T cells. Antiviral T cells reactive against PepTivator SARS-CoV-2 Select could be magnetically enriched from peripheral blood of convalescent COVID-19 patients by small-scale CSA resembling the clinical-grade CCS manufacturing process and showed an activated and cytotoxic T cell phenotype. Four clinical-grade SARS-CoV-2-specific T cell products were successfully generated with sufficient cell numbers and purities comparable to those observed in donor pretesting via CSA. The T cells in the generated products were shown to be capable to replicate, specifically recognize and kill target cells in vitro and secrete cytotoxic molecules upon target recognition. Cell viability, total CD3 + cell number, proliferative capacity and cytotoxic potential remained stable throughout storage of up to 72 h after end of leukapheresis. Conclusion: Clinical-grade SARS-CoV-2-specific T cells are functional, have proliferative capacity and target-specific cytotoxic potential. Their function and phenotype remain stable for several days after enrichment. The adoptive transfer of partially matched, viable human SARS-CoV-2-specific T lymphocytes collected from convalescent individuals may provide the opportunity to support the immune system of COVID-19 patients at risk for severe disease., Competing Interests: OAC reports grants or contracts from Amplyx, Basilea, BMBF, Cidara, DZIF, EU-DG RTD (101037867), F2G, Gilead, Matinas, MedPace, MSD, Mundipharma, Octapharma, Pfizer, and Scynexis; Consulting fees from Amplyx, Biocon, Biosys, Cidara, Da Volterra, Gilead, Matinas, MedPace, Menarini, Molecular Partners, MSG-ERC, Noxxon, Octapharma, PSI, Scynexis, Seres; Honoraria for lectures from Abbott, Al-Jazeera Pharmaceuticals, Astellas, Grupo Biotoscana/United Medical/Knight, Hikma, MedScape, MedUpdate, Merck/MSD, Mylan, Pfizer; Payment for expert testimony from Cidara; Participation on a Data Safety Monitoring Board or Advisory Board from Actelion, Allecra, Cidara, Entasis, IQVIA, Jannsen, MedPace, Paratek, PSI, Shionogi; A patent at the German Patent and Trade Mark Office (DE 10 2021 113 007.7); Other interests from DGHO, DGI, ECMM, ISHAM, MSG-ERC, and Wiley. PK reports grants or contracts from German Federal Ministry of Research and Education (BMBF) B-FAST (Bundesweites Forschungsnetz Angewandte Surveillance und Testung) and NAPKON (Nationales Pandemie Kohorten Netz, German National Pandemic Cohort Network) of the Network University Medicine (NUM) and the State of North Rhine-Westphalia; Consulting fees Ambu GmbH, Gilead Sciences, Mundipharma Resarch Limited, Noxxon N.V. and Pfizer Pharma; Honoraria for lectures from Akademie für Infektionsmedizin e.V., Ambu GmbH, Astellas Pharma, BioRad Laboratories Inc., European Confederation of Medical Mycology, Gilead Sciences, GPR Academy Ruesselsheim, medupdate GmbH, MedMedia, MSD Sharp & Dohme GmbH, Pfizer Pharma GmbH, Scilink Comunicación Científica SC and University Hospital and LMU Munich; Participation on an Advisory Board from Ambu GmbH, Gilead Sciences, Mundipharma Resarch Limited and Pfizer Pharma; A pending patent currently reviewed at the German Patent and Trade Mark Office; Other non-financial interests from Elsevier, Wiley and Taylor & Francis online outside the submitted work. Authors HB, CC, OB, and MS are employed by the company Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany. Author DS is employed by the company Miltenyi Biomedicine GmbH, Bergisch Gladbach, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bonifacius, Tischer-Zimmermann, Santamorena, Mausberg, Schenk, Koch, Barnstorf-Brandes, Gödecke, Martens, Goudeva, Verboom, Wittig, Maecker-Kolhoff, Baurmann, Clark, Brauns, Simon, Lang, Cornely, Hallek, Blasczyk, Seiferling, Köhler and Eiz-Vesper.)

Viral infections and reactivations are major causes of morbidity and mortality after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) as well as in patients with immunodeficiencies. Latent herpesviruses (e.g., cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6), lytic viruses (e.g., adenovirus), and polyomaviruses (e.g., BK virus, JC virus) can cause severe complications. Antiviral drugs form the mainstay of treatment for viral infections and reactivations after transplantation, but they have side effects and cannot achieve complete viral clearance without prior reconstitution of functional antiviral T-cell immunity. The aim of this study was to establish normal ranges for virus-specific T-cell (VST) frequencies in healthy donors. Such data are needed for better interpretation of VST frequencies observed in immunocompromised patients. Therefore, we measured the frequencies of VSTs against 23 viral protein-derived peptide pools from 11 clinically relevant human viruses in blood from healthy donors (n = 151). Specifically, we determined the VST frequencies by interferon-gamma enzyme-linked immunospot assay and classified their distribution according to age and gender to allow for a more specific evaluation and prediction of antiviral immune responses. The reference values established here provide an invaluable tool for immune response evaluation, intensity of therapeutic drugs and treatment decision-making in immunosuppressed patients. This data should make an important contribution to improving the assessment of immune responses in immunocompromised patients., (© 2022. The Author(s).)

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4 th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs (" T cells redirected for universal cytokine-mediated killing "), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD 2 using the CliniMACS Prodigy ® system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD 2 CAR expression and inducible IL-18. Starting with 0.84 x 10 8 and 0.91 x 10 8 T cells after enrichment of CD4 + and CD8 + we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 10 9 and 3.6 x 10 9 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD 2 -CAR mediated activation after co-cultivation with GD 2 -expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD 2 -expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4 + and CD8 + T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD 2 -expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD 2 -expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products., Competing Interests: HA, ASc, and KZ have submitted a patent application describing the “all-in-one” TRUCK vector technology. The University Medicine of Greifswald licensed Ganglidiomab to AnYxis Immuno-oncology for commercialization. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Glienke, Dragon, Zimmermann, Martyniszyn-Eiben, Mertens, Abken, Rossig, Altvater, Aleksandrova, Arseniev, Kloth, Stamopoulou, Moritz, Lode, Siebert, Blasczyk, Goudeva, Schambach, Köhl, Eiz-Vesper and Esser.)

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 7  ± 1.8 × 10 7 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., (© 2021 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background and Aims: Immunocompromised patients are at risk of chronic hepatitis E which can be acquired by blood transfusions. Currently, screening of blood donors (BDs) for HEV RNA with a limit of detection (LOD) of 2,000 IU/ml is required in Germany. However, this may result in up to 440,000 IU of HEV RNA in blood products depending on their plasma volume. We studied the residual risk of transfusion-transmitted (tt) HEV infection when an LOD of 2,000 IU/ml is applied., Methods: Highly sensitive individual donor testing for HEV RNA on the Grifols Procleix Panther system (LOD 7.89 IU/ml) was performed. HEV loads were quantified by real-time PCR., Results: Of 16,236 donors, 31 (0.19%) were HEV RNA positive. Three BDs had viral loads between 710 and 2,000 IU/ml, which pose a significant risk of tt hepatitis E with any type of blood product. Eight BDs had viral loads of >32 to 710 IU/ml, which pose a risk of tt hepatitis E with platelet or plasma transfusions because of their higher plasma volume compared to red blood cell concentrates. Eight of these 11 potentially infectious BDs were seronegative for HEV, indicating a recent infection. Only 8 of 31 donors had viral loads >2,000 IU/ml that would also have been detected by the required screening procedure and 12 had very low HEV loads (<32 IU/ml)., Conclusions: Screening of BDs with an LOD of 2,000 IU/ml reduced the risk of tt HEV infection by about 73% for red blood cell concentrates but by just 42% for platelet and fresh frozen plasma transfusions. Single donor screening (LOD <32 IU/ml) should lead to an almost 100% risk reduction., Lay Summary: Immunocompromised patients, such as solid organ or hematopoietic stem cell recipients, are at risk of chronic hepatitis E, which can be acquired via blood transfusions. The risk of transfusion-transmitted hepatitis E in these patients may not be sufficiently controlled by (mini-)pool hepatitis E virus RNA screening of blood donors. Single donor screening should be considered to improve the safety of blood products., Competing Interests: Conflicts of interest A.H. received a travel grant from Grifols Inc. All other authors declare no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interests All authors declare no conflict of interest regarding this work., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process.

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.

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS ® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 10 8 purified CD3 + T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 10 8 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L + CD3 + T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

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 de novo 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 9 leukocytes collected by lymphapheresis ( n  = 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 6 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 + CD3 + CD8 + cytotoxic T-cells were enriched compared to one process that yielded a higher amount of IFN-γ + CD3 + CD4 + 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.

Several single-nucleotide polymorphisms (SNPs) have been associated with papillary and follicular thyroid cancer (PTC and FTC, respectively) risk, but few have replicated. After analyzing 17525 tag SNPs in 1129 candidate genes, we found associations with PTC risk in SERPINA5, FTO, HEMGN (near FOXE1) and other genes. Here, we report results from a replication effort in a large independent PTC/FTC case-control study conducted in Germany. We evaluated the best tagging SNPs from our previous PTC study and additionally included SNPs in or near FOXE1 and NKX2-1 genes, known susceptibility loci for thyroid cancer. We genotyped 422 PTC and 130 FTC cases and 752 controls recruited from three German clinical centers. We used polytomous logistic regression to simultaneously estimate PTC and FTC associations for 79 SNPs based on log-additive models. We assessed effect modification by body mass index (BMI), gender and age for all SNPs, and selected SNP by SNP interactions. We confirmed associations with PTC and SNPs in FOXE1/HEMGN, SERPINA5 (rs2069974), FTO (rs8047395), EVPL (rs2071194), TICAM1 (rs8120) and SCARB1 (rs11057820) genes. We found associations with SNPs in FOXE1, SERPINA5, FTO, TICAM1 and HSPA6 and FTC. We found two significant interactions between FTO (rs8047395) and BMI (P = 0.0321) and between TICAM1 (rs8120) and FOXE1 (rs10984377) (P = 0.0006). Besides the known associations with FOXE1 SNPs, we confirmed additional PTC SNP associations reported previously. We also found several new associations with FTC risk and noteworthy interactions. We conclude that multiple variants and host factors might interact in complex ways to increase risk of PTC and FTC., (Published by Oxford University Press 2016.)

Granulocyte colony-stimulating factor (G-CSF) is a major regulator of granulopoiesis on engagement with the G-CSF receptor (G-CSFR). The truncated, alternatively spliced, class IV G-CSFR (G-CSFRIV) has been associated with defective differentiation and relapse risk in pediatric acute myeloid leukemia (AML) patients. However, the detailed biological properties of G-CSFRIV in human CD34(+) hematopoietic stem and progenitor cells (HSPCs) and the potential leukemogenic mechanism of this receptor remain poorly understood. In the present study, we observed that G-CSFRIV-overexpressing (G-CSFRIV(+)) HSPCs demonstrated an enhanced proliferative and survival capacity on G-CSF stimulation. Cell cycle analyses showed a higher frequency of G-CSFRIV(+) cells in the S and G2/M phase. Also, apoptosis rates were significantly lower in G-CSFRIV(+) HSPCs. These findings were shown to be associated with a sustained Stat5 activation and elevated miR-155 expression. In addition, G-CSF showed to further induce G-CSFRIV and miR-155 expression of peripheral blood mononuclear cells isolated from AML patients. A Stat5 pharmacological inhibitor or ribonucleic acid (RNA) interference-mediated silencing of the expression of miR-155 abrogated the aberrant proliferative capacity of the G-CSFRIV(+) HSPCs. Hence, the dysregulation of Stat5/miR-155 pathway in the G-CSFRIV(+) HSPCs supports their leukemogenic potential. Specific miRNA silencing or the inhibition of Stat5-associated pathways might contribute to preventing the risk of leukemogenesis in G-CSFRIV(+) HSPCs. This study may promote the development of a personalized effective antileukemia therapy, in particular for the patients exhibiting higher expression levels of G-CSFRIV, and further highlights the necessity of pre-screening the patients for G-CSFR isoforms expression patterns before G-CSF administration.

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.

Integrins are a large family of heterodimeric proteins that are involved in cell adhesion, migration, and proliferation. Integrin diversity and function is regulated by alternative splicing. Membrane-bound and truncated β3-integrins were shown to be key players in cancer metastasis. However, the immunomodulatory functions of the soluble (s) β3-integrin have not been investigated yet. In this study, we described a novel form of sβ3-integrin in acute myeloid leukaemia (AML) patients. Furthermore, we assessed the role of the sβ3-integrin in the modulation of natural killer (NK)-cell activity. Levels of sβ3-integrin were analysed in plasma samples of 23 AML patients and 26 healthy donors by ELISA. The capacity of sβ3-integrin to regulate NK cell activity was investigated using proliferation, cytokine secretion, and cytotoxicity assays. Circulating sβ3-integrin was detected in the plasma of 8 AML patients. NK cells showed significantly higher proliferation rates after stimulation with sβ3-integrin and IL-2, IL-15 (73%). Significant increases in the NK cells' secreted levels of TNF-α, IFN-γ were measured in presence of sβ3-integrin. In addition, sβ3-integrin caused the upregulation of Granzyme B transcripts levels as well as FasL expression levels in NK cells. Most importantly, significantly higher K562 or AML blast target cell lysis rates were observed when NK cells were exposed to sβ3-integrin. This study reports the identification of a novel sβ3-integrin in AML patients and provides novel insights into its role in the immunomodulation of NK cell activity.

Background: There is a correlation between temporal trends of obesity prevalence and papillary thyroid cancer (PTC) incidence in the United States. Obesity is a well-recognized risk factor for many cancers, but there are few studies on the association between obesity and PTC risk. We investigated the association between anthropometric measurements and PTC risk using pooled individual data from three case-control populations., Methods: Height and weight information were obtained from three independent case-control studies, including 1917 patients with PTC (1360 women and 557 men) and 2127 cancer-free controls from the United States, Italy, and Germany. Body mass index (BMI), body fat percentage, and body surface area (BSA) were calculated. An unconditional logistic regression model was used to calculate odds ratios (ORs) and confidence intervals (CIs) with respect to risk of PTC, adjusted by age, sex, race/ethnicity, and study site., Results: In the pooled population, for both men and women, an increased risk of PTC was found to be associated with greater weight, BMI, body fat percentage, and BSA, whereas a reduced risk of PTC was associated with greater height, in the pooled population for both men and women. Compared with normal-weight subjects (BMI 18.5-24.9 kg/m2), the ORs for overweight (BMI 25-29.9 kg/m2) and obese (BMI≥30 kg/m2) subjects were 1.72 [CI 1.48-2.00] and 4.17 [CI 3.41-5.10] respectively. Compared with the lowest quartile of body fat percentage, the ORs for the highest quartile were 3.83 [CI 2.85-5.15] in women and 4.05 [CI 2.67-6.15] in men., Conclusion: Anthropometric factors, especially BMI and body fat percentage, were significantly associated with increased risk of PTC. Future studies of anthropometric factors and PTC that incorporate intermediate factors, including adiposity and hormone biomarkers, are essential to help clarify potential mechanisms of the relationship.

Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4 ng/ml DDAVP. Fluorophor-labeled platelets (10(6)/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320 s(-1)). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells. CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry. G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF. To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.

Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a significant clinical problem. Thus, it would be desirable to have PLT units lacking HLA antigens on the cell surface. Previously, we showed that the generation of functional HLA class I-silenced (HLA-universal) PLTs from CD34(+) cells, using a short hairpin RNA (shRNA) to target β2-microglobulin (β2m) transcripts, is feasible. Here, we assessed the capacity of HLA-silenced PLTs to escape HLA antibody-mediated cytotoxicity in vitro and in vivo. Generation of megakaryocytes (MKs) and PLTs was performed by thrombopoietin-mediated differentiation of HLA-silenced CD34(+) cells within 10 days. Lymphocytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) reporter assays using anti-HLA antibodies and a mouse model for PLT refractoriness were used to assess the immune-evasion capability of HLA-universal MKs and PLTs. To mimic PLT refractoriness in vivo, NOD/SCID/IL-2Rγc(-/-) mice were injected with specific anti-HLA antibodies followed by the infusion of 1 × 10(6) HLA-universal MKs. In vivo PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. Cells expressing a nonspecific shRNA were used as control. Lymphocytotoxicity and ADCC reporter assays showed that HLA silencing protects MKs against HLA antibody-mediated complement-dependent and cell-mediated cytotoxicity. In lymphocytotoxicity assays, 80-90% of HLA-expressing MKs but only 3% of HLA-silenced MKs were lysed. In the circulation of mice, HLA-expressing and HLA-silenced MKs showed PLT production in the absence of anti-HLA antibodies, with human PLT frequencies of up to 0.5% within the PLT population. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, whereas HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and produced PLTs that were detectable up to 11 days. Our data show that HLA-silenced PLTs are efficiently protected against HLA antibody-mediated cytotoxicity and prevent PLT refractoriness in vivo. Provision of HLA-silenced PLTs may become an important component in the management of patients refractory to PLT transfusion.

Adoptive immunotherapy with virus-specific T lymphocytes can efficiently reconstitute antiviral immunity against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) without causing acute toxicity or increasing the risk of graft-versus-host disease. To gain insight into antiviral T cell repertoires and to identify the most efficient antigens for immunotherapy, the frequencies of CMV-, EBV- and ADV-specific T cells in 204 HLA-typed healthy donors were assessed using viral peptides and peptide pools. Confirmatory testing for CMV serology by Western blot technique revealed 19 of 143 (13%) false-positive results. We observed highly significant individual and overall differences in T cell frequencies against CMV, EBV, and ADV antigens, whereas antigen-specific T cells were detected in 100% of CMV- seropositive donors, 73% of EBV- seropositive donors, and 73% of ADV-seropositive donors. At least 124 (61%) potential T cell donors were identified for each virus. Among the tested antigens, frequencies for CMVpp65 and EBVBZLF1 peptide pools were highest. Short-term in vitro peptide stimulation revealed that a donor response to a certain ADV- and EBV-derived peptide may not be determined without prior stimulation. A modified granzyme B ELISpot was used to detect T cell specificity and alloreactivity. Treatment with allogeneic virus-specific cytotoxic T lymphocytes from seropositive third-party donors may be a feasible therapeutic option for infections following cord-blood stem cell transplantation or hematopoietic stem cell transplantation from virus-seronegative donors., (Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)