Fig. 2: Characterization of the SCASA system for CD19 antigen presentation by yeast cells.
From: A yeast surface display platform for characterizing CAR T cell responses to cancer antigens
a Heterologous G protein-coupled receptors (GPCR) as sensory modules for controlling CD19 display with cognate agonists: fold change CD19 levels (top row) and absolute CD19 histograms (lower row). All strains employ PFUS1 and vary between GPCRs and Gɑ-subunits. b Comparison of CD19 levels across GPCRs. c. CD19 SCASA yeast design; CD19 expression is controllable by GPCR-dependent ligands (sensory module). System regulation depends on signaling through an engineered pheromone response pathway (PRP) via Gɑβγ-protein, choice of promoter, and expression boost effects (processing module). CD19 output is a fusion protein composed of HA- and myc-tags, PAS40-linker, (G4S)3-linker, and CD19.1 ECD, fused to Aga2 (effector module). Strains were optimized by; Gɑ-subunit, GPCR, and Aga1 overexpression, as well as gene knock-outs; ste2Δ0, ste3Δ0, gpa1Δ0, sst2Δ0, bar1Δ0, far1Δ0, aga2Δ0. Created in BioRender. Deichmann, M. (2025): https://BioRender.com/dtuelpw. d Relative yEGFP levels of promoters with ɑ-factor stimulation including PRP boost effects, sorted; low to high (5 h. post-induction). e Fold change promoter induction (yEGFP), excluding PRP boost effects (SSC-normalization), sorted; low to high. f Approximated PRP boost of yEGFP expression from quantified PRP activation (black) across all strains and designs (grey) (n = 27). g CD19 histograms of SCASA yeast with different promoters without GPCR stimulation (green), a PPGK1-Empty control lacking CD19 (red), and NALM6 (blue). h Fold change CD19 of SCASA yeast during GPCR stimulation with ɑ-factor (20 h. post-induction). i Approximated PRP boost of CD19 display from quantified PRP activation (black), across all strains and designs (grey) (n = 15). j Comparison of CD19 levels during GPCR stimulation of SCASA yeast, relative to lowest detected CD19 level (top row), and CD19 histograms (lower row). Unless otherwise noted, data is means of median fluorescence intensities (mMFI) for biological replicates (n = 3) and standard deviations hereof. Histograms are representative replicates normalized to the mode. Statistical tests: One- and Two-way ANOVA with multiple comparisons statistical tests. Significance levels: *p ≤ 0.05, **p ≤ 0.001. Not all pairwise comparisons are shown. All statistics and extended analyses in: Supplementary Figs. 1–12 and Supplementary Data 1–6. Source data provided as a Source Data file.
