Fig. 3
From: Precise tuning of gene expression levels in mammalian cells

Fine-tuning antigen expression levels and T-cell activity. a Strategy for tuning Ovalbumin (OVA) expression in B16-F10 melanoma cells using lentivirally integrated miSFITs. Red circles represent SIINFEKL, a peptide antigen derived from ovalbumin. OT-I T-cells express a TCR specific for SIINFEKL presented on MHC-I. b Flow cytometry analysis of OVA-T2A-EGFP expression in B16-F10 cell lines transduced with six different miSFIT variant (miSFITV) lentiviruses (see Supplementary Figure 6 for the gating strategy and distribution of fluorescence intensity) c CD8+, OT-I T-cell activation by OVA-miSFIT B16-F10 cell lines. CD69 expression was quantified by flow-cytometry (n = 5 biological replicates, mean +/− s.d.). d Schematic representation of mixed-culture experimental design. OVA-negative (NGFR-) are mixed with OVA-miSFIT (NGFR+) B16-F10 cells and are challenged overnight with OT-I T-cells. e Representative flow cytometry plots of mixed culture experiments. The percentage of NGFR+ (OVA-miSFIT) cells (blue polygon gate) surviving after overnight selection in the presence or absence of CD8+, OT-I T-cells(at a ratio of 3:1 T-cells to B16-F10 cells) is indicated for each condition. f Relative fitness of B16-F10 cell lines as a function of OVA expression. Relative fitness was calculated by dividing the frequency of NGFR+ cells with T-cells by the frequency of NGFR+ cells without T-cells (n = 3 biological replicates, mean +/− s.d.). Source data are provided as a Source Data file