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Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein.

Crotta S, Stilla A, Wack A, D'Andrea A, Nuti S, D'Oro U, Mosca M, Filliponi F, Brunetto RM, Bonino F, Abrignani S, Valiante NM - J. Exp. Med. (2002)

Bottom Line: This inhibitory effect was observed using both activated and resting NK cells.Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I.These results implicate HCV-E2-mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.

View Article: PubMed Central - PubMed

Affiliation: IRIS, Department of Immunology, Chiron S.p.A., 53100 Siena, Italy.

ABSTRACT
The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in approximately 170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2-mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.

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Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 105 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3[H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).
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fig1: Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 105 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3[H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).

Mentions: Previous results from our group indicated a substantial enrichment of NK cells in HCV-infected livers and their altered expression of KIRs (4, 16). Given the role of CD81 as both an HCV attachment receptor with high affinity for HCV-E2 and a functionally relevant molecule on lymphocytes (9–11), we developed an in vitro model to study the effects of HCV-E2 binding to CD81 on NK cells. We first addressed whether cross-linking NK cell CD81 altered NK cell functions induced by the NK cell triggering receptor CD16 (Fig. 1) . CD16 is the low affinity receptor for IgG (FcγRIII) and is considered the best characterized and most potent stimulatory receptor found on NK cells (7). Using highly purified NK cells obtained from bulk culture, we observed that CD16-induced TNF-α and IFN-γ production (Fig. 1 A and B) was inhibited strongly by coligation of NK cell CD81 with HCV-E2. Similarly, HCV-E2 cross-linking of CD81 blocked CD16-mediated induction of NK cell activation marker (CD25) expression (Fig. 1 C) and cytotoxic granule release (Fig. 1 D). In addition, ligation of CD81 on NK cells substantially inhibited their capacity to proliferate in response to IL-2, demonstrating that the inhibitory effect is not only active on CD16-induced signals (Fig. 1 E). A similar inhibitory effect of CD81 engagement was seen on NK cell IFN-γ production in response to treatment with IL-2, IL-12, or a combination of both (data not shown).


Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein.

Crotta S, Stilla A, Wack A, D'Andrea A, Nuti S, D'Oro U, Mosca M, Filliponi F, Brunetto RM, Bonino F, Abrignani S, Valiante NM - J. Exp. Med. (2002)

Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 105 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3[H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196014&req=5

fig1: Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 105 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3[H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).
Mentions: Previous results from our group indicated a substantial enrichment of NK cells in HCV-infected livers and their altered expression of KIRs (4, 16). Given the role of CD81 as both an HCV attachment receptor with high affinity for HCV-E2 and a functionally relevant molecule on lymphocytes (9–11), we developed an in vitro model to study the effects of HCV-E2 binding to CD81 on NK cells. We first addressed whether cross-linking NK cell CD81 altered NK cell functions induced by the NK cell triggering receptor CD16 (Fig. 1) . CD16 is the low affinity receptor for IgG (FcγRIII) and is considered the best characterized and most potent stimulatory receptor found on NK cells (7). Using highly purified NK cells obtained from bulk culture, we observed that CD16-induced TNF-α and IFN-γ production (Fig. 1 A and B) was inhibited strongly by coligation of NK cell CD81 with HCV-E2. Similarly, HCV-E2 cross-linking of CD81 blocked CD16-mediated induction of NK cell activation marker (CD25) expression (Fig. 1 C) and cytotoxic granule release (Fig. 1 D). In addition, ligation of CD81 on NK cells substantially inhibited their capacity to proliferate in response to IL-2, demonstrating that the inhibitory effect is not only active on CD16-induced signals (Fig. 1 E). A similar inhibitory effect of CD81 engagement was seen on NK cell IFN-γ production in response to treatment with IL-2, IL-12, or a combination of both (data not shown).

Bottom Line: This inhibitory effect was observed using both activated and resting NK cells.Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I.These results implicate HCV-E2-mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.

View Article: PubMed Central - PubMed

Affiliation: IRIS, Department of Immunology, Chiron S.p.A., 53100 Siena, Italy.

ABSTRACT
The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in approximately 170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2-mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.

Show MeSH
Related in: MedlinePlus