The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans

Abstract

Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.

Figure 1.

Soluble NKp30-Fc inhibits NK-92 cytolysis and binds K562 cells. (A) Soluble NKp30-Fc inhibited the killing of K562 cells by NK-92 cells. A 4-h cytoxicity assay was performed in the presence (thin line) or absence (bold line) of 2 µg/ml NKp30-Fc. Data indicate the mean of triplicates ± SD and are representative of three independent experiments. (B) Soluble NKp30-Fc binds K562. The binding of NKp30-Fc to K562 cells was measured by flow cytometry in the presence of blocking anti-NKp30 F(ab′)₂ mAb (AZ20, IgG1) or a negative control anti-NKp46 F(ab′)₂ mAb (BAB281, IgG1). Data are representative of at least three independent experiments.

Figure 2.

Characterization of B7-H6. (A) Direct interaction of B7-H6 with NKp30 using SPR. The data show binding of soluble DKFZp686O24166-Fc to immobilized NKp30-Fc in the presence or absence of blocking anti-NKp30 mAb (1849 and AZ20). Data are representative of three independent experiments. (B) Protein sequence encoded by the B7-H6 gene. Predicted signal peptide, IgV, IgC, and transmembrane domains are indicated. (C) Schematic representation of the B7-H6 and the NKp30 gene orgnaization compared with members of the B7 and the CD28 families, respectively. Each domain is represented as a box and is encoded by exons that are separated by phase 0, 1, or 2 introns as indicated. The scheme refers to the “canonical” sequence as defined by www.uniprot.org. (D) The data show SPR experiments using soluble B7-H1-Fc, B7-H3-Fc, 4IgB7-H3-Fc, B7-H6-Fc, and B7-DC-Fc and immobilized NKp30-Fc. RU, relative units. Data are representative of two independent experiments.

Figure 3.

B7-H6 cell surface expression triggers NKp30-dependent cell activation. (A–C) DO.11.10, DOMSP30, and DOMSP46 cells were stimulated using the indicated plate-bound mAbs (A) or P815.B7-H1 versus P815.B7-H6 cells in the absence (B) or presence of indicated F(ab′)₂ mAbs or B7-H6 mouse antiserum (C). Data indicate the mean of triplicates + SD and are representative of three independent experiments. (D and E) NK-92 cells (D) and IL-2–stimulated primary NK cells (E) were used in a cytolytic assay against P815 cells (thin line) or P815.B7-H6 cells (bold line). Soluble NKp30-Fc (thin dashed line) and control Fc-fusion protein (B7-H3-Fc; bold dashed line) were used at 2 µg/ml. (F) Freshly isolated primary blood NK cells in PBMCs were tested for their activation induced by P815.B7-H1 or P815.B7-H6 cells in the presence or absence of anti-NKp30 F(ab′)₂ mAbs or control mouse Ig (mIg). 500,000 PBMCs were added to 100,000 tumor cells. The fraction of reactive NK cells (percentage of responding NK cells) was assessed by adding the percentage of CD107⁺IFN-γ⁻, CD107⁺IFN-γ⁺, and CD107⁻IFN-γ⁺ NK cells. Data are representative of three independent experiments. Error bars indicate SD. Statistical analysis was performed using a Mann-Whitney U test. *, P < 0.05.

Figure 4.

B7-H6 is not detected on blood cells but is expressed on tumor cells. (A and B) Assessment of B7-H6 cell surface staining on PBMCs, P815 cell transfectants, and tumor cells was performed by flow cytometry using anti–B7-H6 mouse antiserum. (C) NKp30-Fc cell surface staining on tumor cells is also represented. HIg, control human Ig; mIg, control mouse Ig. (D) Freshly isolated primary blood NK cells in PBMCs were tested for their activation induced by the indicated cell lines in the presence or absence of anti-NKp30 F(ab′)₂ mAbs, B7-H6 mouse antiserum, or control mouse Ig (mIg). 500,000 PBMCs were added to 100,000 tumor cells. The fraction of reactive NK cells (percentage of responding NK cells) was assessed by adding the percentage of CD107⁺IFN-γ⁻, CD107⁺IFN-γ⁺, and CD107⁻IFN-γ⁺ NK cells (mean + SD; n = 3 independent experiments). Statistical analysis was performed using a Mann-Whitney U test. *, P < 0.05. Open histograms, B7-H6 staining; filled histograms, control mIg staining.