Impact of polyclonal anti-CD3/CD28-coated magnetic bead expansion methods on T cell proliferation, differentiation and function

Background and aimsThe 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.MethodsPeripheral 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.ResultsAn 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).ConclusionsAn 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.

► We compared different T cell expansion assays with anti-CD3/CD28-coated magnetic bead.
► An equal bead-to-cell ratio is optimal in promoting proliferations and preserving cellular vitality.
► A high bead-to-cell ratio favored the selection of bioactive T cells.
► The combination of homeostatic cytokines further improved cell output.