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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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CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (107 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (107 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).
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fig4: CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (107 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (107 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).

Mentions: The activation of protein tyrosine kinases (PTKs) and their phosphorylation of diverse substrates is a necessary event in NK cell activation through CD16 and other NK triggering receptors (21, 22). Therefore, we examined the capacity of CD81 cross-linking to block this critical signaling pathway. Analysis of total PTK activity was performed by determining the extent of phosphotyrosine (p-Tyr) substrates induced in NK cells by CD16 cross-linking alone or in combination with anti-CD81 or anti-CD56 treatment. The overall p-Tyr levels induced through CD16 were substantially diminished after simultaneous ligation of CD81 (Fig. 4 A). The examination of specific PTK substrates known to be phosphorylated in NK cells as a result of CD16 stimulation and important for CD16-induced functions yielded similar results (Fig. 4 B and C). Specifically, we chose for this analysis: (i) CD3ζ which is directly associated with CD16 in NK cells and whose phosphorylation is induced immediately proximal to CD16 ligation (23); and (ii) erk-2 that is part of the more downstream MAPK cascade important for NK cell cytokine gene expression and proliferation (24). The tyrosine phosphorylation of both signaling moieties was specifically inhibited after CD81 ligation. In addition, cross-linking with the anti-CD81 or anti-CD56 reagents alone had no effect on the extent of phosphorylated proteins detected in the NK cells in all of the experiments.


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)

CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (107 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (107 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).
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Related In: Results  -  Collection

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fig4: CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (107 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (107 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).
Mentions: The activation of protein tyrosine kinases (PTKs) and their phosphorylation of diverse substrates is a necessary event in NK cell activation through CD16 and other NK triggering receptors (21, 22). Therefore, we examined the capacity of CD81 cross-linking to block this critical signaling pathway. Analysis of total PTK activity was performed by determining the extent of phosphotyrosine (p-Tyr) substrates induced in NK cells by CD16 cross-linking alone or in combination with anti-CD81 or anti-CD56 treatment. The overall p-Tyr levels induced through CD16 were substantially diminished after simultaneous ligation of CD81 (Fig. 4 A). The examination of specific PTK substrates known to be phosphorylated in NK cells as a result of CD16 stimulation and important for CD16-induced functions yielded similar results (Fig. 4 B and C). Specifically, we chose for this analysis: (i) CD3ζ which is directly associated with CD16 in NK cells and whose phosphorylation is induced immediately proximal to CD16 ligation (23); and (ii) erk-2 that is part of the more downstream MAPK cascade important for NK cell cytokine gene expression and proliferation (24). The tyrosine phosphorylation of both signaling moieties was specifically inhibited after CD81 ligation. In addition, cross-linking with the anti-CD81 or anti-CD56 reagents alone had no effect on the extent of phosphorylated proteins detected in the NK cells in all of the experiments.

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