Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity

Abstract

Circulating human natural killer (NK) lymphocytes have been functionally defined by their ability to exert cytotoxic activity against MHC class I-negative target cell lines, including K562. Therefore, it was proposed that NK cells recognized the "missing self." We show here that the Ig-like CD160 receptor expressed by circulating CD56(dim+) NK cells or IL-2-deprived NK cell lines is mainly involved in their cytotoxic activity against K562 target cells. Further, we report that HLA-C molecules that are constitutively expressed by K562 trigger NK cell lysis through CD160 receptor engagement. In addition, we demonstrate, with recombinant soluble HLA-Cw3 and CD160 proteins, direct interaction of these molecules. We also find that CD158b inhibitory receptors partially interfere with CD160-mediated cytotoxicity, whereas CD94CD159a and CD85j have no effect on engagement with their respective ligands. Thus, CD160HLA-C interaction constitutes a unique pathway to trigger NK cell cytotoxic activity.

Fig 1.

Activation of fresh peripheral blood NK cell-mediated cytolysis by CD160. (A) Cytotoxic activity of freshly isolated human PB-NK separated in CD160⁺ and -160⁻ populations against K562 target cell in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 (CD160). (B) Cytotoxic activity of PB-NK either untreated (CD160^bright+) or cultured for 3 days with IL-2 (CD160^dim+) against K562 target cell in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 or Ig-isotype control (IgG1). (C) CD160 expression in fresh PB-NK cells either untreated (−IL-2) or cultured for 3 days with IL-2 (+IL-2) was analyzed by flow cytometry using FITC-labeled BY55 mAb. Data are representative of several independent experiments. Open profile is Ig-isotype control staining. (D) Redirected lysis assay of P815 using fresh human CD160⁺ PB-NK in the presence of mAbs specific for the indicated molecules or control IgG1. The effector-to-target cell (E:T) ratio used was 25. Results of A, B, and D are mean ± SD of triplicates and are representative of three independent experiments.

Fig 2.

CD160 engagement triggers cytolytic activity in NKL, NK92, and NK3.3 cell lines. Shown is the cytotoxicity of NKL, NK92, and NK3.3 effector cells cultured without IL-2 against K562 in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 (CD160) or control IgG1. Results are mean ± SD of triplicates and are representative of three independent experiments.

Fig 3.

Engagement of CD160 by HLA-C, constitutively expressed by K562, triggers NK cell cytotoxicity. (A) K562 cells were analyzed by flow cytometry for surface expression of HLA class I using broad anti-HLA-class I (mAb W6/32), anti-HLA-C (mAb B1.23.2), anti-HLA-C free heavy chains (mAb L31), anti-HLA-E (mAb MEM-E/06), or anti-HLA-G (mAb 87G) (black profiles) followed by PE-labeled conjugate. Open profiles are Ig-isotype control stainings. Data are representative of several independent experiments. (B) Cytotoxicity of NK92 cultured without IL-2 against K562 in the absence of mAb (no mAb) or in the presence of mAb MEM-E/06 (HLA-E, 5 μg/ml), mAb B1.23.2 (HLA-C, 10 μg/ml), or Ig-isotype control (IgG). Results are mean ± SD of triplicates and are representative of several independent experiments.

Fig 4.

Soluble recombinant HLA-Cw3 and CD160 proteins bind to JAR-Cw3 and Jurkat-CD160, respectively. (A Upper) By flow cytometry, mAb CL1-R2 stained Jurkat-CD160 but not Jurkat cells (black profiles). Open profiles are Ig-isotype control stainings. (A Lower) flow cytometry binding of soluble HLA-Cw3 cross-linked with B1.23.2 mAb, followed by incubation with PE-streptavidin on Jurkat-CD160 and NK.3.3 cells but not Jurkat (black profiles). Open profiles are control staining with B1.23.2 mAb and streptavidin-PE. (B) Flow cytometry showing binding of mAb B1.23.2 on JAR-Cw3 but not JAR (Upper, open profiles are Ig-isotype control staining), and of soluble CD160-Flag, after incubation with anti-Flag mAb followed by PE-labeled conjugate on Jurkat-CD160 and NK.3.3 cells but not Jurkat (Lower, open profiles are staining after incubation with control Jurkat supernatant). (C) Cytotoxicity of NK92 cultured without IL-2 against JAR-Cw3 target cells in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 (CD160). Results are mean ± SD of triplicates and are representative of three independent experiments.

Fig 5.

CD160-induced NK92 cytotoxicity is not blocked by inhibitory receptors specific for HLA-E and -G, whereas HLA-C, through its interaction with CD158b, alters CD160-induced NK3.3 cytotoxicity. (A) Surface expression of HLA class I on K562-G transfectants was monitored with W6/32 (broad HLA class I), B1.23.2 (HLA-C), L31 (free HLA-C heavy chains), MEM-E/06 (HLA-E), or 87G (HLA-G) mAbs (black profiles). Open profiles are Ig-isotype control stainings. Data are representative of several independent experiments. (B) Cytotoxicity of NK92 cultured without IL-2 against K562-G target cells, either in the absence of mAb (no mAb) or in the presence of isotype control (IgG), mAb B1.23.2 (HLA-C), mAb 87G (HLA-G), or mAb MEM-E/06 (HLA-E). The effector-to-target cell (E:T) ratio was 10. Results are mean ± SD of triplicates and are representative of several independent experiments. (C) Redirected lysis assay of P815, using NK92 cells in the presence of anti-CD160 or -CD59 mAbs mixed with anti-CD159a or -CD85j mAb or with control IgG. The E:T ratio used was 25. Results are mean ± SD of triplicates and are representative of three independent experiments. (D) Cytotoxicity of NK3.3 cultured without IL-2 against K562 in the absence of mAb (no mAb) or in the presence of mAb B1.23.2 (HLA-C), or (E) mAb CL1-R2 (CD160), anti-CD158b (CD158b) mAb, or anti-CD158b together with anti-CD160 (CD160/CD158b) mAbs. Results are mean ± SD of triplicates and are representative of three independent experiments.