Fig 2: High peptide selectivity, cross-reactive binding and functional inhibition of KIR2DL3+ natural killer (NK) cells by HLA-C*05:01 (C2). (A) Specific lysis of 221–C*05:01–ICP47 cells at different E:T by YTS-2DL3 NK cells. 221–C*05:01–ICP47 target cells were used after overnight culture at 26°C in the presence of no peptide (NP), P2, P2-AA, P2-EE, P2-GG, and P7. (B) Relative-specific lysis of 221–C*05:01–ICP47 cells by YTS-2DL3 NK cells compared to NP at10:1 (E:T). Mean and SEM of three independent experiments are shown. *** = p < 0.001 by Student’s t-test. (C) Flow cytometry histograms showing degranulation (CD107a expression) of KIR2DL3-SP (left) and KIR3DL1-SP (right) NK cells in response to 221–C*05:01–ICP47 cells loaded with P2, P2-AA, P2-EE, or NP. (D) Box plots showing the fraction of CD107a+ KIR2DL3-SP NK cells in response to 221–C*05:01–ICP47 cells loaded with P2, P2-AA, P2-EE, P2-GG, P2-LA, P11, P11-7A, P10, P10-AA, P10-7E, or NP (n = 3–5). The CD107a+ values were normalized to the fraction of CD107a+ KIR2DL3-SP NK cells that degranulated in the absence of peptide (set at 1.0) for each donor (CD107a+ ranged from 62.8 to 79.0%, n = 5). ***p < 0.001, **p < 0.01, and *p < 0.05 by Kruskal–Wallis test with Dunn’s multiple comparisons test. (E) KIR2DL3-Fc binding to 221–C*05:01–ICP47 cells correlated with degranulation (CD107a+) of KIR2DL3-SP NK cells in response to 221–C*05:01–ICP47 cells in the presence of P2, P2-AA, P2-EE, P2-GG, P2-LA, P11, P11-7A, P10, P10-AA, P10-7E, or NP. Spearman’s correlation r = −0.955, p < 0.0001. CD107a expression was normalized to value obtained in response to NP for each donor.

Fig 3: KIR2DL2 and KIR2DL3 are more peptide selective than KIR2DL1 but less stringent about allotype preference. KIR2DL1, with strong specificity for C2 allotype, exhibits low peptide selectivity in binding C2. KIR2DL2 and KIR2DL3 cross-reactivity with C2 is associated with greater peptide selectivity. KIR2DL1 cross-reacted with C1 in the context of only two peptides. This hierarchy in peptide selectivity of killer cell Ig-like receptors (KIR)–HLA-C interactions is relevant to natural killer (NK) biology and understanding of disease associations. Peptide sequence-driven binding of KIR to HLA-C provides a potential mechanism for pathogen as well as self-peptide to modulate NK cell function through altering levels of KIR binding and KIR-mediated inhibition.

Fig 4: Human leukocyte antigen (HLA)-C*05:01 (group C2) and HLA-C*08:02 (group C1) are almost identical in sequence and HLA-C*05:01-eluted endogenous peptides bind HLA-C*05:01 and HLA-C*08:02. (A) Schematic showing how the specificity of inhibitory KIR for different HLA-C allotypes is defined by an amino acid dimorphism at positions 77 and 80 of HLA-C, where KIR2DL1 binds group C2 allotypes (Asn77Lys80) and KIR2DL2 and KIR2DL3 bind group C1 allotypes (Ser77Asn80). (B) Nucleotide sequence alignment of amino acid positions 77–80 of HLA-C*05:01 and HLA-C*08:02. Bold indicates nucleotide differences. (C) The motif for HLA-C*05:01 9mer peptides. (D) HLA-I expression on 221–C*05:01–ICP47 (left) and RMA-S-C*08:02 (right) cells after overnight incubation at 26°C with 100 μM HLA-C*05:01 peptides P2, P10, and P11 and no peptide (NP). (E) HLA-I expression on 221–C*05:01–ICP47 (left) and RMA-S-C*08:02 (right) cells after overnight incubation at 26°C with 100 μM of 1 of 28 HLA-C*05:01 peptides and NP. Mean MFI and SEM of three independent experiments are shown.

Fig 5: KIR2DL2 and KIR2DL3 binding to human leukocyte antigen (HLA)-C*08:02 is more peptide selective than KIR2DL1 binding to HLA-C*05:01. (A) KIR2DL1-Fc binding to peptide-loaded 221–C*05:01–ICP47 (left), KIR2DL2-Fc (middle) and KIR2DL3-Fc (right) binding to RMA-S-C*08:02 cells. Cells were loaded overnight at 26°C with no peptide (NP) or 100 μM HLA-C*05:01 peptides P2, P4, and P7. (B) Same as in panel (A). Killer cell Ig-like receptor (KIR-Fc) binding was assessed after overnight incubation at 26°C with NP or 100 μM of 28 individual HLA-C*05:01 peptides. Mean MFI and SEM of three independent experiments are shown. KIR-Fc (3.6 μg/ml) was conjugated to protein A-alexa 647.