Figure 5.

Sequence-function map elucidating which CD86 mutants escape MIR2 downregulation by comparing the frequency of population III and IV as illustrated inFigure 2, A and B. The WT sequence at each position is indicated by a white square with a gray dot. Red squares indicate variants that are protected from MARCH1-mediated downregulation, whereas white to blue squares indicate variants that are downregulated equally well or better than WT CD86. Gray squares indicate variants that are lost from the library during the selection process. Diagonal lines within each square represent standard errors, with longer lines indicating noisier data. A colourized CD86 model is shown where the shade of each residue is the average hue of the squares in the black-boxed region of the sequence-function map. Residues throughout the TM domain of CD86 protect from MIR2-mediated downregulation but are not clustered on any one face of the helix. Mutations selected for follow-up studies are boxed in green, and results are shown in Figure 6. MIR2, modulator of immune recognition 2; TM, transmembrane.