Transcription factor cooperativity impacts CD16-synNotch performance. (a)–(c) Optimal values of (a) lncSHP-coding plasmid, (b) cytolytic molecule-coding plasmid, and (c) initial synNotch for negative cooperativity (lightest shade), no cooperativity (intermediate shade), and positive cooperativity (darkest shade). (d)–(f) The amount of secreted GZMB from CD16-synNotch pair when using the optimal values from (d) round 1, (e) round 3, and (f) round 6 from (a)–(c). (g)–(i) The amount of secreted PRF1 from CD16-synNotch pair when using the optimal values from (g) round 1, (h) round 3, and (i) round 6 from (a)–(c). (d)–(i) Markers, mean model prediction; error bars, one standard deviation; lightest shade, negative cooperativity ( 0.5); intermediate shade, no cooperativity ( 1); darkest shade, positive cooperativity ( 1.5).