Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions

Abstract

Infection with hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide. Little is known about how this virus is able to persist or whether this persistence might be because of its ability to alter the early innate immune response. The major HCV envelope protein E2 has been shown to bind to CD81. Thus, HCV binding to natural killer (NK) cells could result in the cross-linking of CD81. To explore this possibility, we investigated whether cross-linking CD81 on NK cells could alter NK cell function. CD81 cross-linking by monoclonal antibody (mAb) specific for CD81 or by immobilized E2 have been shown to result in costimulatory signals for human T cells. In this study, we show that CD81 cross-linking via immobilized E2 or mAbs specific for CD81 inhibits not only non major histocompatibility complex-restricted cytotoxicity mediated by NK cells but also interferon (IFN)-gamma production by NK cells after exposure to interleukin (IL)-2, IL-12, IL-15, or CD16 cross-linking. These results show that CD81 cross-linking mediates completely different signals in NK cells versus T cells. Importantly, these results suggest that one mechanism whereby HCV can alter host defenses and innate immunity is via the early inhibition of IFN-gamma production by NK cells.

Figure 1.

Cross-linking of CD81 significantly inhibits IL-2–mediated IFN-γ production by NK cells. Purified NK cells (10⁵ cells per well) were cultured in microtiter plates that were precoated with anti-CD81 (5 μg/ml), anti-CD56 (5 μg/ml), immobilized E2 (anti-E2 (5 μg/ml)/E2 (1 μg/ml) complex), or anti-E2 (5 μg/ml). Each culture contained 200 μl of medium with or without supplemented IL-2 (100 U/ml). Supernatants were collected after 18 h and assessed for IFN-γ levels by ELISA. The amounts of IFN-γ are presented as mean (pg/ml) ± SD of duplicate samples. Data are representative of five independent experiments using different donors.

Figure 2.

Cross-linking of CD81 significantly inhibits IL-12– or IL-15–mediated IFN-γ production by NK cells. Purified NK cells (10⁵ cells per well) were cultured in microtiter plates that were precoated with control IgG1 5 μg/ml antibody, 5 μg/ml anti-CD81, 5 μg/ml anti-E2, or immobilized E2 (5 μg/ml anti-E2/1 μg/ml E2 complex). Each culture contained 200 μl of medium supplemented with either IL-12 (500 U/ml) or IL-15 (5 U/ml). Supernatants were collected after 18 h and assessed for IFN-γ levels by ELISA. The amounts of IFN-γ are presented as mean (pg/ml) ± SD of duplicate samples. Data are representative of three independent experiments using different donors.

Figure 3.

Differential effects of CD81 crosslinking on the activation of NK cells versus TCR-γδ⁺ or TCR-αβ⁺ T cells. (A) Purified NK cells, TCR-γδ⁺ T cells, or TCR-αβ⁺ T cells were cultured in wells (10⁵ per well) precoated with following antibodies: (i) 0.5 μg/ml anti-CD16 (NK cells) or anti-CD3 (T cells); (ii) anti-CD16 plus anti-E2 (5 μg/ml) for NK cells or anti-CD3 plus anti-E2 for T cells; or (iii) anti-CD16 plus immobilized E2 (1 μg/ml anti-E2/E2) for NK cells, or anti-CD3 plus immobilized E2 (anti-E2/E2) for T cells. TCR-γδ⁺ T cells were derived from bacteria-expanded cultures, as described in Materials and Methods. TCR-αβ⁺ T cells were derived from anti–CD3- or ConA-activated PBL. Each culture contained 200 μl of medium supplemented with IL-2 (100 U/ml). Supernatants were collected after 18 h and assessed for the IFN-γ levels by ELISA. IFN-γ levels are presented as mean (pg/ml) ± SD of duplicate samples. Data are representative of two independent experiments using different cell preparations. (B) Purified NK cells or TCR-γδ⁺ T cells were cultured in wells precoated with varying concentrations of antibody specific for CD81. For NK cells, IL-2 (100 U/ml) was added to each well. For T cells, wells were precoated with varying concentrations of anti-CD81 along with a constant amount of anti-CD3 (0.5 μg/ml). Culture supernatants were collected after 18 h and assessed for IFN-γ levels by ELISA. IFN-γ levels are presented as mean (pg/ml) ± SD of duplicate samples. Data presented is derived from one of two independent experiments. (*P < 0.05; **P < 0.01; Student's t test).

Figure 3.

Differential effects of CD81 crosslinking on the activation of NK cells versus TCR-γδ⁺ or TCR-αβ⁺ T cells. (A) Purified NK cells, TCR-γδ⁺ T cells, or TCR-αβ⁺ T cells were cultured in wells (10⁵ per well) precoated with following antibodies: (i) 0.5 μg/ml anti-CD16 (NK cells) or anti-CD3 (T cells); (ii) anti-CD16 plus anti-E2 (5 μg/ml) for NK cells or anti-CD3 plus anti-E2 for T cells; or (iii) anti-CD16 plus immobilized E2 (1 μg/ml anti-E2/E2) for NK cells, or anti-CD3 plus immobilized E2 (anti-E2/E2) for T cells. TCR-γδ⁺ T cells were derived from bacteria-expanded cultures, as described in Materials and Methods. TCR-αβ⁺ T cells were derived from anti–CD3- or ConA-activated PBL. Each culture contained 200 μl of medium supplemented with IL-2 (100 U/ml). Supernatants were collected after 18 h and assessed for the IFN-γ levels by ELISA. IFN-γ levels are presented as mean (pg/ml) ± SD of duplicate samples. Data are representative of two independent experiments using different cell preparations. (B) Purified NK cells or TCR-γδ⁺ T cells were cultured in wells precoated with varying concentrations of antibody specific for CD81. For NK cells, IL-2 (100 U/ml) was added to each well. For T cells, wells were precoated with varying concentrations of anti-CD81 along with a constant amount of anti-CD3 (0.5 μg/ml). Culture supernatants were collected after 18 h and assessed for IFN-γ levels by ELISA. IFN-γ levels are presented as mean (pg/ml) ± SD of duplicate samples. Data presented is derived from one of two independent experiments. (*P < 0.05; **P < 0.01; Student's t test).

Figure 4.

CD81 inhibits IL2 activation of NK cells and TCR-γδ⁺ T cells. Purified NK cells or TCR-γδ T cells were cultured in wells (10⁵ cells per well) either untreated or precoated with control IgG1 antibody (5 μg/ml) or anti-CD81 (5 μg/ml). Each culture contained 200 μl of medium with or without supplemented IL-2 (100 U/ml). Supernatants were collected after 18 h and assessed for IFN-γ levels by ELISA. The levels of IFN-γ are presented as mean (pg/ml) ± SD of duplicate samples. Data are representative of three independent experiments using different donors.

Figure 5.

CD81 cross-linking inhibits the cytotoxic activities of fresh NK cells. The standard 4-h chromium release assay was used to assess NK cell–mediated cytotoxicity. Varying numbers of freshly isolated NK cells were cultured in microtiter wells precoated control IgG1, anti-CD81, or anti-CD56 antibody at 1 μg/ml of each antibody, 5 μg/ml anti-E2, or immobilized E2 (1 μg/ml anti-E2/E2) for 30 min before addition of 51Cr-labeled K-562 (10⁴ cells per well). Effector:target cell ratios of 20, 10, 5, and 2.5 were used in each experiment. Spontaneous releases for K-562 were always below 10%. Specific lysis is presented as mean percentage ± SD of duplicate samples. Data are representative of six independent experiments using different cell preparations.