Supplementary Figure 2: Generation and testing of KIRζ⁺ Jurkat reporter cell lines and assessment of HLA class I OCs on target cells.

(a) Staining of anti–pan-HLA class I complex was assessed in Jurkat cells that underwent CRISPR/Cas9-mediated β₂m knockout (KO). (b) KIRζ chimeric receptors for inhibitory and activating KIRs were designed by fusing the extracellular domain (ECD) and transmembrane domain (TMD) of the indicated KIRs with the triple immunoreceptor tyrosine-based activating motif (ITAM)-containing cytoplasmic tail (CYT) of CD3ζ. (c) KIR expression was assessed in Jurkat-β₂m-KO cells stably transduced with the indicated KIRζ chimeric receptors via staining with anti–KIR2DL2-KIR2DL3, anti–KIR3DS1-KIR3DL1, and anti–KIR3DL1-KIR3DL2-KIR3DS1; UT signifies untransduced. (d) Reporter activity of untransduced (UT), KIR3DL1ζ⁺ and KIR3DS1^hiζ⁺ Jurkat cells co-incubated with cell-sized beads covalently conjugated to irrelevant mouse IgG (mIgG) or anti–KIR3DS1-KIR3DL1 antibody (Z27) was measured as percentage of CD69^hi Jurkat cells. (e) Untransduced (UT), KIR2DL3ζ⁺, KIR3DL1ζ⁺, KIR3DS1^hiζ⁺ and KIR3DL2ζ⁺ Jurkat cells were co-incubated with streptavidin beads coated with free biotin (negative control; NC) or biotinylated HLA-F monomers (HLA-F) and the percentage of bead-binding cells (left panel) as well as reporter activity as a percentage of CD69^hi cells (right panel) was measured. (f) Staining of anti–pan-HLA class I complex (clone: W6/32) and an antibody to HLA class I OCs (clone: HC10) antibodies was measured on untreated and acid-pulsed cell lines to confirm OC generation; 2° only signifies secondary only staining. Data in d is representative of three experiments and shows three technical replicates; a, c, e, and f show one experiment; d shows mean + s.d.