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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 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ+ T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR+ T (stippled bar), CD16+ T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR+ T; Th1; CD16+ T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16+ T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).
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fig3: CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ+ T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR+ T (stippled bar), CD16+ T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR+ T; Th1; CD16+ T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16+ T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).

Mentions: NK cells and T cells share many phenotypic and functional features (17). Small subsets of T cells also express characteristic NK cell markers such as CD16 and CD56. In addition, KIR+ T cells can be isolated from the blood of healthy individuals and these T cells are inhibited by KIR recognition of HLA class I ligands, similar to what is routinely observed for NK cells (18). A previous report from Wack et al. demonstrated that bulk populations of T cells were costimulated by CD81 cross-linking, however this study did not investigate T cells bearing NK cell receptors (11). Therefore, we generated T and NK cell clones from healthy individuals and compared their responses after CD81 engagement (Fig. 3) . Similar to our previous results with cultured or resting NK cells, the NK cell clones were all inhibited by CD81 cross-linking either with an anti-CD81 mAb or HCV-E2 (Fig. 3 A). By contrast, none of the T cell clones tested, representing six distinct phenotypic/functional subsets, were inhibited after simultaneous ligation of CD81 and CD3 (Fig. 3 B and C). In fact, 5 out of the 6 subsets tested (TCR αβ+ “classical”; KIR+; NKT; Th1; and Th2 T cell clones) were all costimulated by simultaneous CD81 and CD3 engagement similar to previous results using bulk populations of resting T cells and PHA-activated T cell lines (11). The one exception was found using a rare and understudied population of T cells bearing the characteristic NK marker, CD16 (19). Unlike NK cells and other T cells, the response of these cells to cocross-linking of CD3 and CD81 on their surface was neutral. This failure of CD81 ligation to elicit any response in CD16+ T cells was observed when we measured CD3-induced IFN-γ (Fig. 3 C) and TNF-α production, as well as, CD3-mediated proliferation and activation marker expression (data not shown). Ligation of CD16 on these T cell clones had no effect, consistent with a previous report (20).


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 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ+ T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR+ T (stippled bar), CD16+ T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR+ T; Th1; CD16+ T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16+ T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).
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fig3: CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ+ T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR+ T (stippled bar), CD16+ T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR+ T; Th1; CD16+ T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16+ T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).
Mentions: NK cells and T cells share many phenotypic and functional features (17). Small subsets of T cells also express characteristic NK cell markers such as CD16 and CD56. In addition, KIR+ T cells can be isolated from the blood of healthy individuals and these T cells are inhibited by KIR recognition of HLA class I ligands, similar to what is routinely observed for NK cells (18). A previous report from Wack et al. demonstrated that bulk populations of T cells were costimulated by CD81 cross-linking, however this study did not investigate T cells bearing NK cell receptors (11). Therefore, we generated T and NK cell clones from healthy individuals and compared their responses after CD81 engagement (Fig. 3) . Similar to our previous results with cultured or resting NK cells, the NK cell clones were all inhibited by CD81 cross-linking either with an anti-CD81 mAb or HCV-E2 (Fig. 3 A). By contrast, none of the T cell clones tested, representing six distinct phenotypic/functional subsets, were inhibited after simultaneous ligation of CD81 and CD3 (Fig. 3 B and C). In fact, 5 out of the 6 subsets tested (TCR αβ+ “classical”; KIR+; NKT; Th1; and Th2 T cell clones) were all costimulated by simultaneous CD81 and CD3 engagement similar to previous results using bulk populations of resting T cells and PHA-activated T cell lines (11). The one exception was found using a rare and understudied population of T cells bearing the characteristic NK marker, CD16 (19). Unlike NK cells and other T cells, the response of these cells to cocross-linking of CD3 and CD81 on their surface was neutral. This failure of CD81 ligation to elicit any response in CD16+ T cells was observed when we measured CD3-induced IFN-γ (Fig. 3 C) and TNF-α production, as well as, CD3-mediated proliferation and activation marker expression (data not shown). Ligation of CD16 on these T cell clones had no effect, consistent with a previous report (20).

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