Optimised Antibody Free Magnetic Cell Sorting of primary human CD4+ T cells.

<p>Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.