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

Tseng CT, Klimpel GR - J. Exp. Med. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.

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.

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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).
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fig3: 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).

Mentions: CD16 is a low-affinity IgG receptor (FcγRIII) that is expressed primarily on NK cells (23). Cross-linking of CD16 on NK cells has been shown to activate NK cells with regards to enhanced cytokine production and cytolytic activity (24). To further investigate the inhibitory actions of CD81 cross-linking on NK cell functions, we investigated whether CD81 cross-linking also inhibited NK cell activation via CD16 cross-linking. Since the cross-linking of CD81 on T cells results in an enhanced proliferative response and an enhancement in cytokine production after CD3 cross-linking (18–20), NK cells and T cells appear to differ significantly with regards to the consequences of CD81 cross-linking. To verify and further explore this interesting difference, we assessed T cells and NK cells in parallel for the effects of CD81 cross-linking following the cross-linking of CD3 on T cells versus CD16 on NK cells. To address this question, NK cells, TCR-γδ+ T cells, and TCR-αβ+ T cells were exposed to varying combinations of immobilized E2 or anti-CD81 and assessed for IFN-γ production at 18 h of culture. As shown in Fig. 3 A and B the simultaneous cross-linking of CD81 and CD3 resulted in an enhanced production of IFN-γ by TCR-αβ+ and TCR-γδ+ T cells. These results are identical to previously published findings (18–20). In striking contrast, when CD81 was cross-linked along with CD16 cross-linking (Fig. 3 A) or IL-2 stimulation (Fig. 3 B) a significant inhibition of IFN-γ production by NK cells was observed. As seen in Fig. 4 , these differences were not due to potential differences in the stimuli used for activation of NK cells versus T cells since identical results were also observed when both TCR-γδ+ T cells and NK cells were assessed for IFN-γ production after IL-2 stimulation. Cross-linking CD81 on T cells or NK cells with either immobilized E2 or mAb specific for CD81 yielded equivalent results. These results show that instead of providing costimulatory signals, as seen with T cells, CD81 cross-linking on NK cells induces negative regulatory signals that inhibit or downregulate NK cell activation.


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

Tseng CT, Klimpel GR - J. Exp. Med. (2002)

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).
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fig3: 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).
Mentions: CD16 is a low-affinity IgG receptor (FcγRIII) that is expressed primarily on NK cells (23). Cross-linking of CD16 on NK cells has been shown to activate NK cells with regards to enhanced cytokine production and cytolytic activity (24). To further investigate the inhibitory actions of CD81 cross-linking on NK cell functions, we investigated whether CD81 cross-linking also inhibited NK cell activation via CD16 cross-linking. Since the cross-linking of CD81 on T cells results in an enhanced proliferative response and an enhancement in cytokine production after CD3 cross-linking (18–20), NK cells and T cells appear to differ significantly with regards to the consequences of CD81 cross-linking. To verify and further explore this interesting difference, we assessed T cells and NK cells in parallel for the effects of CD81 cross-linking following the cross-linking of CD3 on T cells versus CD16 on NK cells. To address this question, NK cells, TCR-γδ+ T cells, and TCR-αβ+ T cells were exposed to varying combinations of immobilized E2 or anti-CD81 and assessed for IFN-γ production at 18 h of culture. As shown in Fig. 3 A and B the simultaneous cross-linking of CD81 and CD3 resulted in an enhanced production of IFN-γ by TCR-αβ+ and TCR-γδ+ T cells. These results are identical to previously published findings (18–20). In striking contrast, when CD81 was cross-linked along with CD16 cross-linking (Fig. 3 A) or IL-2 stimulation (Fig. 3 B) a significant inhibition of IFN-γ production by NK cells was observed. As seen in Fig. 4 , these differences were not due to potential differences in the stimuli used for activation of NK cells versus T cells since identical results were also observed when both TCR-γδ+ T cells and NK cells were assessed for IFN-γ production after IL-2 stimulation. Cross-linking CD81 on T cells or NK cells with either immobilized E2 or mAb specific for CD81 yielded equivalent results. These results show that instead of providing costimulatory signals, as seen with T cells, CD81 cross-linking on NK cells induces negative regulatory signals that inhibit or downregulate NK cell activation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.

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.

Show MeSH
Related in: MedlinePlus