title: Comparative Analysis of Clinical-Scale IFN-γ-Positive T-Cell Enrichment Using Partially and Fully Integrated Platforms.
abstract: OBJECTIVE
  The infusion of enriched CMV-specific donor T-cells appears to be a suitable alternative for the treatment of drug-resistant CMV reactivation orde novoinfection 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 × 109leukocytes 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 × 106viable 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%).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Priesner
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Esser
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School , Niedersachsen , Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Tischer
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Marburger
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School , Niedersachsen , Germany.
authors.forename: Krasimira
authors.initials: K
authors.lastname: Aleksandrova
authors.affiliation: Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany; Department of Pediatric Hematology and Oncology, Hannover Medical School, Niedersachsen, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Maecker-Kolhoff
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School , Niedersachsen , Germany.
authors.forename: Hans-Gert
authors.initials: HG
authors.lastname: Heuft
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School , Niedersachsen , Germany.
authors.forename: Lilia
authors.initials: L
authors.lastname: Goudeva
authors.affiliation: Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany; Institute for Transfusion Medicine, Hannover Medical School, Niedersachsen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.
authors.forename: Lubomir
authors.initials: L
authors.lastname: Arseniev
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Köhl
authors.affiliation: Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany; Institute for Transfusion Medicine, Hannover Medical School, Niedersachsen, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Eiz-Vesper
authors.affiliation: Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Klöß
keywords: CliniMACS Cytokine-Capture-System; closed GMP-compliant systems; immunoaffinity cell selection; single platform and multicolor flow cytometry; virus-specific T-cells