Fig 1: Screening and functional validation of KIR2DS4-binding peptides with open-HLA-C*04:01. (A) KIR2DL1-Fc, KIR2DS4-Fc, and HLA-I mAb binding to 221-C*04:01-ICP47 cells preloaded with peptides #1, K8E, and four “self” peptides containing p8 Tyr. Source proteins for the “self” peptides are shown. (B) KIR-Fc (KIR2DL1, KIR2DL3, KIR2DS1, and KIR2DS4) binding to open-HLA-C*04:01 exchanged with VV9 p7 (VYDVRQxYV) and p8 (VYDVRQAxV) library peptides. Binding was measured by ELISA. (C) Data from (B) shown as violin plots with VV9 (open squares) and VV9-substituted ligands (filled circles). (D) KIR2DS4-Fc and β2M antibody binding to open-HLA-C*04:01 displayed on CombiCells and exchanged with selected VV9 peptides. (E and F) Flow-cytometric gating strategy used to identify the KIR2DS4+ KIR2DL1− NK cell population and response to CombiCells displaying open-HLA-C*04:01 loaded with VV9-7S or VV9-7W (F). (G) Degranulation of KIR2DS4+ KIR2DL1− cells as in (F), following exposure to CombiCells displaying HLA-C*05:01-P2-MW or open-HLA-C*04:01 loaded with indicated peptides. Data from three independent experiments using NK cells from three separate donors are shown.
Fig 2: KIR binding to peptide-exchanged HLA-C*05:01. (A and B) KIR-Fc binding (KIR2DL1, KIR2DS1, KIR2DS4) binding to open-HLA-C*05:01 with placeholder peptide P2-EE or exchanged to peptides with indicated residues at p7p8. Open-HLA-C*05:01 was conjugated to plates in (A) and CombiCells in (B) via SpyCatcher. KIR-Fc binding was measured by ELISA in (A) and flow cytometry in (B), with level of conjugation displayed by anti-β2m mAb. (C) KIR-Fc binding to HLA-C*05:01(P2-FV) after dipeptide exchange (GV or GL), followed by exchange with peptides P2-MW or P2-IA. KIR-Fc binding was measured by ELISA.
Fig 3: Recognition of peptide-exchanged HLA-C*05:01 by primary KIR2DL1+ NK cells. (A) Display of ULBP1 and CD155 on CombiCells detected by mAbs and NKG2D-Fc, and DNAM-1-Fc, respectively. (B and C) KIR2DL1+ and KIR2DL1− NK-cell degranulation (CD107a upregulation) in response to ULBP1, CD155, and HLA-C*05:01 displayed on CombiCells. (C) Three types of HLA-C*05:01 were tested, all containing P2 (IIDKSGSTV); wild-type, open, and dipeptide exchanged. Data from three independent experiments with NK cells from separate donors are shown.
Fig 4: Development and validation of a peptide-exchange strategy for HLA-C*04:01. (A) SDS–PAGE of purified inclusion bodies (IBs) from open-C*04:01 heavy chain (G120C) and β2M (H31C (left). Size-exclusion chromatography (SEC) trace from purification of refolded open-HLA-C*04:01 complex (middle). SDS-PAGE of refolded open-HLA-C*04:01 complexes loaded with peptide #1 or K8E, run under nonreducing and reducing conditions (right). (B) KIR2DL1-Fc binding to open-HLA-C*04:01 displayed on CombiCells following indicated peptide exchange. (C) Peptide-binding motifs of HLA-C*04:01 and -C*05:01 (top). Amino acid frequency at position 2 for HLA-C*04:01 and HLA-C*05:01 (bottom). (D) KIR2DL1-Fc binding to open-HLA-C*04:01 displayed on CombiCells loaded with placeholder P2-EE or after exchange to #1. #1 = QYDDAVYKL, K8E = QYDDAVYEL, P2-EE = IIDKSGEEV.
Supplier Page from R&D Systems, a Bio-Techne Brand for KIR2DL1/CD158a Fc Chimera Protein
Available conjugates: Sizes Available: 50 ug