title: Impact of polyclonal anti-CD3/CD28-coated magnetic bead expansion methods on T cell proliferation, differentiation and function.
abstract: OBJECTIVE
  The aim of the present study was to compare several in vitro anti-CD3/CD28-coated magnetic bead expansion methods as a means of improving T cell recovery and to evaluate the impact of the respective methods on T cell viability, differentiation and function. The effect of the respective expansion protocols on cytokine production and cytotoxicity was also characterized.
  
  
  METHODS
  Peripheral blood mononuclear cells were isolated from healthy donors, expanded for 7 days by different methods in vitro and then counted. T cell viability, phenotype and differentiation status were determined by flow cytometry. The cytotoxicity of collected cells was evaluated by a non-radioactive assay.
  
  
  RESULTS
  An equal bead-to-cell ratio in the presence of IL-2, IL-7 and IL-15 generated the highest T cell yields (median 4.75-fold increase [range 4.10-6.25], P=0.043) with a median of 79.20% viable cells (range 70.00%-80.30%). By contrast, a high bead-to-cell ratio (3:1) favored the selection of central memory T cells (CD4: 0.44 [0.40-1.69]; CD8: 0.77 [0.42-1.19], P=0.043) with increased interferon-gamma (IFN-γ) production following re-stimulation (48.5% [42.1%-68.2%], P=0.043) and also a trend towards enhanced cytotoxicity against target cells (10:1 ratio: 61.80% [40.80%-80.00%], P=0.068).
  
  
  CONCLUSIONS
  An equal bead-to-cell ratio is optimal in keeping the balance between promoting proliferations and preserving cellular vitality and the combination of homeostatic cytokines further improved cell output, whereas a high bead-to-cell ratio favored the production of bioactive cells.
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shi
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Tianhong
authors.initials: T
authors.lastname: Wan
authors.forename: Yanning
authors.initials: Y
authors.lastname: Liu
authors.forename: Guoping
authors.initials: G
authors.lastname: Peng
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhu
mesh_terms: D000906:Antibodies / Q000494:pharmacology*; D018106:CD28 Antigens / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D060328:Magnetic Phenomena; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects
chemical_list: D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D016207:Cytokines