PMC

(A) The constitutive expression of MICA/B but not ULBPs in HeLa cells, as assessed by flow cytometry. The representative results of three independent experiments are shown. (B) HeLa cells were labeled with CytoTrack green and then co-cultured with PBMC with HNK1/HNK (n = 3) and LNK/LNK (n = 3) for 4 and 16 h. The frequency of cell death is shown. (C) The representative results showing cell death in HeLa cells cultured with PBMCs from an individual with HNK/HNK or LNK/LNK genotype for 16 h, as assessed by flow cytometry. (D) NK cells degranulation (CD3−, CD56+, CD107+) after PBMCs from individuals with HNK/HNK (3) or LNK/LNK (3) genotype were co-cultured with HeLa cells for 16 h and assessed by flow cytometry. The data are shown as the standard deviation ± fold-change (% CD107 without target vs. % CD107 with target).

(A) Representative flow cytometry histogram indicating higher NKG2D expression in NK cells with the HNK/HNK genotype. (B) Freshly isolated NK cells from a cohort of 42 healthy Japanese individuals with HNK/HNK (n = 4), LNK/HNK (n = 21) and LNK/LNK (n = 17) genotypes were examined for NKG2D expression at the cell surface by flow cytometry. (C) NK samples derived from a cohort of 33 healthy Japanese individuals were assessed for NKG2D expression as in B. The NKG2D expression in NK cells (D) and CD8+ T cells (E) in a cohort of 20 Vietnamese healthy individuals was assessed by flow cytometry. The MFI values for NKG2D expression are indicated in (B,C,D and E) with each dot representing one individual.

(A) The MICA/B expression on the surface of OUN1 cells with (blue histogram) or without VPA treatment (orange histogram), as assessed by flow cytometry, (red histogram; isotype stained cells). The representative results of three independent experiments are shown. (B) The cytotoxicity of HNK1/HNK (n = 6) and LNK/LNK (n = 13) NK cells against OUN-1 cells with or without VPA treatment (expressing NKG2D-L) in the presence or absence of a blocking anti-NKG2D mAb, as determined by a ⁵¹Cr release assay. (C) The expression of ULBP3 in uninfected (orange histogram) or CMV infected (blue histogram) HEL cells as assessed by flow cytometry (D) Cytotoxicity of HNK1/HNK (n = 6) and LNK/LNK (n = 13) NK cells against CMV-infected human fibroblasts in the presence or absence of a blocking anti-NKG2D mAb. (E) IFN-γ secreted by PBMC from individuals with the HNK1/HNK genotype (n = 3) and LNK/LNK (n = 7) cultured with or without NKG2D-Ls and assessed by an ELISA assay.

PBMCs from individuals with HNK/HNK (3) or LNK/LNK (3) genotype were cultured for four days in IL-2 containing medium in the presence of either recombinant MICA (NKG2D-L) or TGFβ1 (10 ng/ml) and the expression of NKG2D receptor on NK cells and CD8+ T cells was assessed by flow cytometry. (A) The gating strategy used to assess NKG2D expression in NK cells and CD8+ T cells. (B) A representative histogram figure showing NKG2D expression in control (dark histogram), rMICA treated, (blue histogram) or TGFβ1 treated (red histogram) NK cells is shown in the upper panel. The bottom panel shows summarized results from different individuals with HNK1/HNK (n = 3) and LNK/LNK (n = 3) and the data represent the NKG2D intensity (% MFI) in treated cells compared with that in the control cells. (C) A representative histogram figure showing NKG2D expression in control (dark histogram), rMICA treated, (blue histogram) or TGFβ1 treated (red histogram) CD8+ T cells is shown in the upper panel. The bottom panel shows summarized results from different individuals with HNK1/HNK (n = 3) and LNK/LNK (n = 3) and the data represent the NKG2D intensity (% MFI) in treated cells compared with that in the control cells.

(A) Allele-specific transcriptional activity showing allelic imbalance. The HNK allele had higher transcriptional activity than the LNK allele in cDNA samples but not in gDNA samples from individuals heterozygous for the rs1049174 polymorphism. (B) Modulation of reporter gene expression by NKG2D 3′UTR polymorphism. YT cells and HEK cells were transfected with a luciferase expression vector pGL3 TK, or with constructs including the NKG2D 3′UTR HNK1-allele (pGL3 TK HNK1) or the LNK1-allele (pGL3 TK LNK1). The insertion of NKG2D 3′UTR segments repressed luciferase activity. The LNK allele construct, however, repressed the luciferase activity to a significantly greater degree in the YT transfected cells than in the miR1245-lacking HEK cells. (C) The rs1049174 SNP 3′UTR region of NKG2D occurs in the miR-1245 binding site, and miR-1245 is expressed in NK cells. A schematic representation of the location 1049174 SNP within the miR-1245 target site in the 3′UTR region of NKG2D. The presence of the HNK genotype (1163C) interrupts the pair-complementarity, decreasing the affinity between NKG2D mRNA and miR-1245. (D) The ectopic induction of miR1245 expression in HEK cells resulted in greater luciferase activity repression induced by the LNK allele. HEK293 cells were co-transfected with miR-1245 and pGL3 TK-Luc HNK or pGL3 TK-Luc vectors. At 48 h after transfection, the luciferase activity was measured. Firefly luciferase activity was normalized to renilla luciferase expression, and the means ± the standard error of the percentage of activities in the control situation (cells transfected with luciferase plasmid without NKG2D 3′UTR) are shown.