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Keywords:

  • Cell activation;
  • Cytoskeleton;
  • NK cells;
  • T cells;
  • Tetraspanin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

The hepatitis C virus (HCV) binds to human cells through the interaction of its envelope glycoprotein E2 with the tetraspanin CD81. We have previously reported that engagement of CD81 has opposite effects on T and NK cell function, as it enhances T cell receptor-mediated T cell activation and inhibits CD16- or IL-12-mediated NK cell activation. We further investigated this dichotomy and found that another tetraspanin, CD82, induces the same opposing effects on human primary T and NK cells. Activation by other unrelated stimuli such as NKG2D- and beta-1 integrin is also reduced by CD81 ligation on NK cells. CD81 engagement by monoclonal antibody or HCV-E2 enhances zeta and Erk phosphorylation in T cells and reduces them in NK cells, reflecting the opposite functional outcomes. CD81 engagement induces dramatic morphological changes and local F-actin accumulation in both NK and T cells, indicating rearrangement of the actin cytoskeleton. Pharmacological inhibition of actin polymerization reduces T cell activation, whereas it greatly enhances NK cell activation. Importantly, treatment with actin blockers abolishes the inhibitory effect of CD81 ligation on NK cells. We propose that tetraspanin engagement leads to comparable cytoskeleton reorganization in T and NK cells, which in turn results in opposite functional outcomes.

Abbreviations:
CCD:

Cytochalasin D

HCV:

Hepatitis C virus

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

CD81 is a member of the tetraspanin family, an evolutionarily conserved set of cell surface proteins containing four membrane spanning domains, two extracellular loops and short N- and C-terminal cytoplasmic domains. Tetraspanins are generally regarded as molecular linkers, connecting not only extracellular and cytoplasmatic elements but also laterally other tetraspanin family members into a specific, highly organized network, the tetraspanin web 1, 2, which includes other membrane-associated proteins and may serve as a scaffold for signaling processes 3. The lateral associations of tetraspanins extend to integrins, mainly of the β1 type 46, members of the G-protein-coupled receptors (GPCR 7), the CD19-CD21 complex on B cells 8 and CD4 on T cells 9. No associated proteins have been described on NK cells.

In addition to lateral associations within the same membrane, tetraspanins also function as receptors for secreted or membrane-bound proteins and for proteins derived from infectious disease-causing organisms 1. In particular, CD81 is required for Plasmodium sporozoite entry into hepatocytes 10 and for the entry of the hepatitis C virus (HCV) into human cells 1113, demonstrating that CD81 is a cellular receptor for the virus. In fact, CD81 had been previously shown to bind the HCV envelope protein E2 14, 15.

Although tetraspanins have no recognizable motifs for the docking of signaling molecules, they associate with intracellular signaling components. They can recruit activated PKC, which is involved in integrin phosphorylation 16, and the intracellular component of the GPCR, the G protein subunit Gα 7. In non-immune cells, tetraspanins CD9, CD63, CD151 and CD81 have been shown to associate with phosphatidylinositol 4-kinase (PtdIns 4-K), a key enzyme in the synthesis of phosphoinositides 17, 18.

Engagement of tetraspanins on cells of the immune system leads in many instances to enhanced activation, as in the case of B cells where CD81 co-ligation is thought to recruit the CD19-CD21 costimulatory complex to the BCR 3. In addition, appropriate engagement of CD81 alone on B cells leads to activation (Rosa et al, submitted for publication). On T cells, co-engagement of CD9, CD53, CD63, CD81 or CD82 with CD3 has a strong co-stimulatory effect 1923, but the underlying mechanism is less clear than in B cells. While associations between CD81 and CD4 have been demonstrated, costimulation is found also in CD8 T cells, in CD4-CD8- double negative γδT cells and in Jurkat cells with very low expression of CD4 22, 24, 25. In the Jurkat T cell line, CD81 has been directly related to Lck-dependent T cell responses 24, and CD82 cross-linking was shown to induce phosphorylation and association of Vav-1 and SLP76, indicating that Rho-GTPases participate in CD82 signaling 26. The only indications of negative effects of tetraspanin engagement on immune cells are reduced FcϵR-triggered degranulation of mast cells upon CD81 engagement 27 and increased T cell activity in mice deficient for Tssc6 28.

Others and we have previously demonstrated that CD81 ligation by HCV-E2 has dramatic effects on cells of the immune system, costimulating T cells and blocking NK cell activities such as cytokine production, cytotoxicity and IL-2-driven proliferation 22, 29, 30. These findings defined an efficient HCV immune evasion strategy through the inhibition of the early antiviral activities of NK cells, which may contribute to the high efficiency of the virus in establishing chronic infections. Moreover, the costimulatory effects of HCV-E2 on T cells may contribute to immune-mediated liver damage and autoimmune extrahepatic manifestations often associated with HCV infection.

In this study, we investigate the mechanism mediating the effects of CD81 engagement. CD81 and other tetraspanins have been described to compartmentalize in lipid rafts in different cell lines, including Jurkat T cells 24, 31, 32. Lipid rafts, defined by their relative insolubility in non-ionic detergents, are highly ordered lateral assemblies of glycosphingolipids and cholesterol that provide a microenvironment favorable to protein tyrosine kinase-coupled signaling cascades 33. As ligand-induced clustering of elemental rafts to form larger and more stable domains has been proposed as a key step in promoting the signaling process 34, 35, we investigated the possibility that the raft-related localization of CD81 differs in T and NK cells which would explain the opposite responses of these cell types to tetraspanin-engagement.

Cytoskeleton rearrangements play a central role in the assembly of signaling components that are required in lymphocyte activation 3638. Tetraspanins have been implicated in many processes that involve regulation of the cytoskeleton, namely migration, adhesion and cell polarization 39. CD82 appears to be a link between the cell membrane and the cytoskeleton, as CD82 engagement leads to its specific association with the cytoskeleton 40 and induces morphological changes that are dependent on Rho-GTPase activity and involve VAV and SLP76 activation 26. In addition, CD81 and CD82 have been found in the contact zone between APC and T cells where the T cell receptor and signaling molecules assemble in a cytoskeleton-dependent manner 41, 42. Therefore, we compared CD81-mediated effects on the cytoskeletal rearrangement in primary T and NK cells and analyzed the outcome of actin re-organization on T and NK cell function.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

CD81 provides a global ‘off’ signal for NK cells

In previous studies we demonstrated that cross-linking of CD81 by HCV-E2 or anti-CD81 antibodies reduced NK cell activation induced by either cytokines or CD16 29, 30. To extend these observations, we asked whether other activation stimuli were affected by cross-linking of CD81. Using highly purified NK cells obtained from bulk cultures, we found that IFN-γ (Fig. 1A, B) and TNF-α production (not shown) induced by the engagement of β1 integrin or the NKG2D receptor were strongly inhibited by co-engagement of CD81 with plastic-immobilized HCV-E2 or anti-CD81 antibody, at levels comparable to the effect of CD81 on CD16- mediated activation (Fig. 1C). In contrast, T cells were not inhibited but rather costimulated by CD81 engagement (Fig. 1D). We conclude that a wide range of stimuli can be inhibited by CD81-engagement on NK cells.

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Figure 1. Cross-linking of tetraspanins by HCV-E2 or antibodies on NK cells blocks cytokine production induced by different stimuli. NK cells (A, B, C and E) or T cells (D and F) were stimulated for 24 h, and the supernatants were analyzed by ELISA for IFN-γ (A-D) or TNF-α (E, F) production. For these experiments, NK cells were cultured in the presence of anti-NKG2D, anti-β1integrin or anti-CD16 antibody alone (□) or in combination with 10 μg/mL of: anti-CD56 (▵); anti-HCV E2 (○); anti-HCV E2 + rHCV-E2 (•); anti-CD81 (♦) or anti-CD82 (▴). T cells were cultured in the presence of anti-CD3 alone (□) or in combination with 10 μg/mL of: anti-HLA class I (▵); anti-HCV-E2 (○); anti-HCV-E2 + rHCV-E2 (•); anti-CD81 (♦); anti-CD82 (▴). Experiments to determine the optimal concentration of anti-CD81, anti-CD82 or anti-HCV-E2 + rHCV-E2 demonstrated that their effects were detectable over a broad range of concentrations (0.2–20 μg/mL) (data not shown). Each of these experiments, as all others in this paper, was repeated at least 4 times with similar results.

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Ligation of the tetraspanin CD82 has effects similar to CD81 engagement on T and NK cells

Among tetraspanins expressed on both T and NK cells, CD82 has been shown to costimulate Jurkat T lymphoma cells 19. We decided to assess whether CD82 mediates the same opposite effects as CD81 on these lymphocyte subpopulations.

Purified T and NK cells were stimulated with mAb to CD3 and CD16, respectively, in the presence or absence of anti-CD82 antibody. As shown in Fig. 1E and F, cross-linking with an anti-CD82 mAb inhibited CD16-induced TNF-α production by NK cells, while T cells were costimulated by simultaneous CD3 and CD82 engagement. Similar results were found for IFN-γ production (not shown). Thus, the ability to mediate opposing effects on T and NK cells can be extended to tetraspanins other than CD81.

Biochemical responses to CD81 ligation

On NK cells, engagement of CD16 causes the activation of protein tyrosine kinases and phosphorylation of diverse substrates 43, 44. We demonstrated previously that CD81 cross-linking could block this signaling pathway, causing a reduction in the overall level of tyrosine phosphorylation induced by CD16 triggering. More specifically, both the ζ chain, which is directly associated to CD16, and the more downstream MAPK Erk-2 are phosphorylated to a lesser extent as a consequence of CD81 cross-linking 29.

To investigate the mechanism underlying the opposing effects of CD81 engagement on NK cells and T cells, we performed similar experiments on T cells. As shown in Fig. 2A, CD3 cross-linking induced a dose-dependent tyrosine phosphorylation of Erk 1/2 MAPK. The concomitant engagement of CD81 provided a costimulus for the CD3 signal, leading to Erk1/2 phosphorylation even in the presence of a sub-optimal anti-CD3 stimulus. Importantly, co-engagement of CD81 on primary T cells with recombinant HCV-E2 protein led to the same co-stimulatory effect on the CD3-induced signal as that observed with anti-CD81 mAb (Fig. 2B).

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Figure 2. CD81 engagement increases phosphorylation of downstream targets of TCR signaling. (A) T cells were incubated with the indicated antibodies and cross-linked with GαM for 3 minutes at 37°. Cellular lysates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, immunoblotted with a rabbit polyclonal anti-phospho-p44/42 MAPK (p-Erk) antibody and then reprobed with a rabbit polyclonal p44/42 MAPK (Erk) antibody. (B) Plates were coated with anti-CD3 alone or in combination with HCV-E2 bound to plastic via anti-HCV-E2 mAb (10 μg/mL). Cells were activated for 10 minutes at 37°C on coated plates, then collected and lysed. Lysates were tested for Erk phosphorylation as described above.

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These data represent a biochemical correlate of the divergent functional outcome of CD81 ligation on NK and T cells, demonstrating that CD81 engagement has opposite effects on the signaling pathways downstream of CD3 on T cells and of CD16 on NK cells.

CD81 and raft clustering

As we could not ascribe to the engagement of CD81 the induction of any specific biochemical pathway, it may be that CD81 engagement influences the overall ability to deliver downstream signals. As raft microdomains are known to facilitate signaling processes in lymphocytes 45, 46, we assessed the localization of CD81 in rafts in NK and T cells.

Purified NK cells and T cells were treated with 1% Triton X-100 and the lysates loaded onto a sucrose gradient to separate detergent resistant and detergent soluble fractions, which were subsequently analyzed by immunoblot. As shown in Fig. 3A, comparable amounts of CD81 were present in the detergent insoluble light fractions of both NK and T cells. The adapter protein LAT, which in unstimulated cells is distributed along the whole gradient but peaks in the low-density raft fractions, was used as a loading control to compare the two raft preparations.

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Figure 3. CD81 localizes to lipid rafts in both NK and T cells. (A) Purified NK or T cells were lysed in 1% Triton X-100 and subjected to sucrose density gradient centrifugation. Gradient fractions were collected and analyzed by SDS-PAGE, followed by immunoblot for CD81 and LAT. Lanes were loaded with 30 μL (lanes 2–6) or 10 μL (lanes 7–9) of each fraction. (B) NK or T cells were cultured for 24 h in plates coated respectively with the indicated concentration of anti-CD16 or anti-CD3 alone (□) or in combination with: cholera toxin B (CtxB) bound to plastic via rabbit anti-CtxB serum (•), or anti-CD81 (♦), 10 μg/mL. Anti-CtxB serum alone or CtxB in solution had no effect (not shown).

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In addition, we induced aggregation of lipid rafts by cross-linking of the raft resident GM1 using cholera toxin B (CtxB) subunit and anti-CtxB antibodies. We found that forcing the clustering of lipid rafts enhanced the production of IFN-γ (Fig. 3B) and TNF-α (not shown) by both T and NK cells. CtxB in solution did not influence T or NK cell activation (not shown).

These data indicate that CD81 is partially resident in rafts in both T and NK cells and that CD81 effects are not merely due to raft coalescence, which enhances both CD3- and CD16-induced signals.

CD81 engagement induces cytoskeleton rearrangements in both T cells and NK cells

Signaling through the tetraspanin CD82 triggers its association with the cytoskeleton sustaining morphological changes and activation of Jurkat T cells 40. Therefore, we assessed whether CD81 has similar effects on primary cells. Purified T and NK cells were incubated on plates coated with anti-CD81 with or without an additional stimulus (PMA or anti-CD3/anti-CD16). Upon CD81 cross-linking, both T and NK cells showed an altered morphology characterized by single cells with long dendrite-like filopodia, and cells did not form the characteristic clumps induced by most types of activation (Fig. 4A, B, arrowheads). Anti-CD81 alone induced some degree of morphological modification, but these became more dramatic in the presence of PMA or anti-CD3/anti-CD16 (Fig. 4A, B). Immobilized control antibodies (CD56 for NK cells and MHC class I for T cells), used alone or in combination with the above stimuli, did not influence morphological changes (not shown). These data suggest that tetraspanin engagement induces similar cytoskeleton reorganization in NK and T cells. Cytoskeleton rearrangement was also evidenced by F-actin capping induced by receptor cross-linking. Cells were incubated with the indicated antibodies, plated onto plastic-immobilized secondary antibodies, and F-actin capping was assessed after staining of fixed cells with TRITC-phalloidin (Fig. 5). On both T and NK cells, engagement of CD81 induced F-actin capping comparable to that caused by CD3- or CD16-stimulation, respectively. F-actin capping was enhanced on both T and NK cells when co-engaged together with CD16 or CD3.

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Figure 4. Both T and NK cells show morphological changes upon CD81 cross-linking. T cells (A) or NK cells (B) were plated onto microculture plates coated with the Abs indicated and incubated at 37°C. Where indicated, PMA (20 ng/mL) was added at the beginning of the culture. After 6 h, cells were photographed on a bright light microscope.

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Figure 5. CD81 co-cross-linking enhances F-actin capping on T and NK cells. Cells were pre-incubated on ice with the indicated Abs and stimulated on goat anti-mouse-Ab coated plates for 1 h at 37°C. Cells were then removed, fixed, permeabilized, stained for actin with TRITC-phalloidin and analyzed under a confocal microscope. Micrographs show examples of cells without or with actin capping, the bar chart indicates the percentages of cells found with F-actin caps. 150 cells were counted for each treatment.

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In conclusion, the effects of CD81 cross-linking on cytoskeleton rearrangement are comparable in T and NK cells.

Opposite effect of cytoskeleton block on T andNK cell activation

To investigate how cytoskeleton rearrangement is linked to cellular activation, T and NK cells were treated prior to activation with low doses of cytochalasin D (CCD), an inhibitor of actin polymerization. CD3-triggered TNF-α production by T cells is reduced by preincubation with CCD, whereas TNF-α production by NK cells is greatly enhanced (Fig. 6A, B). This surprising increase of NK cell activity was also observed when IFN-γ production was assessed (not shown). As expected, higher doses of CCD treatment lead to inhibition of both T and NK cells, and we did not find any low dose of CCD that would lead to enhanced T cell activation (not shown). After CCD treatment, we did not observe the morphological changes usually found after cell activation, but cells remained rounded, indicating that CCD in fact inhibits cytoskeleton reorganization at these low concentrations (not shown). To exclude CCD specific side effects, experiments were repeated using the structurally unrelated molecules Latrunculin A and B. Similar effects on TNF-α and IFN-γ production by T and NK cells were observed when using these blockers of actin polymerization at concentrations between 3 μM and 0.3 μM (Fig. 6C, D and data not shown). We conclude that inhibition of cytoskeleton rearrangement has a negative impact on T cell activation and an enhancing effect on NK cell activation.

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Figure 6. Impaired actin polymerization reduces T cell and enhances NK cell activation. T cells (A, C) or NK cells (B, D, E) were preincubated for 30 minutes at 37°C in cytochalasin D (CCD) (A, B). In (C-E), cells were preincubated with 1 μM Latrunculin A (Lat A), Latrunculin B (Lat B) or CCD. Cells were subsequently cultured in the plates coated with antibodies as annotated (CD3, CD16, CD81) or in the presence of the indicated concentrations of IL-12 in solution (E). After 24 h, supernatants were collected and tested by ELISA for cytokine production. In (C-E), the level of cytokine production by non-pretreated, activated cells is indicated by a horizontal bar for comparison. The use of DMSO carrier alone did not have any effect on T or NK cell activity.

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To assess if tetraspanins and actin blockers exert similar modulations on other activation pathways, we tested their effect on IL-12-induced IFN-γ production. We could not test TNF-α production, as IL-12 is a poor inducer of this cytokine in NK cells. Fig. 6E shows that engagement of CD81 reduces and pretreatment with F-actin blockers enhances IFN-γ production by NK cells in response to IL-12. Thus, two NK activation mechanisms, as unrelated as CD16 triggering and IL-12 addition, are modulated by tetraspanins and cytokine blockers in a similar manner.

It has been previously shown that the requirement of cytoskeleton rearrangement for T cell activation depends on the type of stimulus. In particular, activation in a T cell-APC system requires actin rearrangement, as CCD blocks activation, whilst the use of soluble anti-CD3 cross-linked by soluble goat-anti-mouse Ab is not blocked by CCD 36. To assess the cytoskeleton requirements of T cell and NK cell activation by solid- or fluid-phase stimuli, we directly compared the effect of actin blockers on activation through plastic-immobilized mAb or mAb cross-linked in solution. As shown in Fig. 7, activation of T cells by fluid-phase stimuli is not blocked by a range of CCD concentrations and is therefore largely independent from cytoskeleton rearrangement. In contrast, CCD leads to enhanced cytokine production by NK cells stimulated by both fluid- and solid-phase stimuli, indicating that the cytoskeleton can negatively regulate both types of NK cell activation.

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Figure 7. Block of cytoskeleton rearrangement enhances NK cell activation by both solid- and fluid-phase stimuli. Purified T or NK cells were preincubated without or with CCD (3μM) and stimulated (A) in plates coated with anti-CD3 (1μg) or anti-CD16 (1μg), respectively, or (B) by soluble anti-CD3 (10μg) or anti-CD16 (10μg), cross-linked by goat-anti mouse F(ab)2 fragments (30μg/mL). After 24 h, TNF-α secretion was tested by ELISA. Neither uncrosslinked anti-CD3, anti-CD16 nor goat-anti mouse Abs alone induced significant activation (not shown). For better comparison, the amounts of TNF-α produced in the absence of CCD were set as 100%. Similar results were found for CCD concentrations ranging from 10μM to 1μM and for production of IFN-γ (not shown).

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To see if the CD81-mediated inhibitory effects on NK cells are dependent on cytoskeleton rearrangements, we assessed TNF-α production by cells pretreated with different concentrations of CCD and subsequently activated by cross-linking of CD16 alone or in combination with CD81. Fig. 8A shows that in the absence of CCD, CD81 co-engagement leads to a substantial reduction of TNF-α production. Increasing doses of CCD reduced and eventually abolished this inhibitory effect of CD81 engagement on cytokine production. Fig. 8B shows the same data normalized to TNF-α production induced by CD16 alone: in the absence of CCD, the residual activity after cross-linking of CD16 and CD81 is about 20% of that induced by anti-CD16 alone, whereas the residual activity at 1 μM CCD is 51% and at 3 μM CCD 128%. Altogether, our findings suggest that CD81 triggered inhibition of NK cells is mediated by actin rearrangement.

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Figure 8. The inhibitory effect of CD81 engagement on NK cells depends on intact cytoskeleton. (A) Purified NK cells were preincubated with CCD and stimulated in plates coated with anti-CD16 with or without anti-CD81, as indicated. After 24 h, TNF-α secretion was tested by ELISA. (B) shows the relative amounts of TNF-α induced by CD16 and CD81 engagement when normalized to those induced by engagement of CD16 alone. Comparable effects were seen over a broad range of anti-CD16 concentrations.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

In this study, we show that engagement of CD81 or other tetraspanins on NK cells inhibits activation triggered by signals as diverse as NKG2D, β1 integrin, CD16 or IL-12. We also describe the biochemical events downstream of CD81 engagement on T and NK cells, which correlate with the opposite functional outcomes in these two lymphocyte populations.

Dramatic morphological changes and F-actin capping are induced by CD81 engagement in both T and NK cells, indicating the ability of CD81 to induce cytoskeleton rearrangements. The pharmacological block of actin polymerization has opposite effects on these cells, leading to reduced T cell activation and increased NK cell activation. In addition, the same actin blockers can abolish the inhibitory effect of CD81 on NK cells. Taken together, these observations suggest that tetraspanin-induced cytoskeleton rearrangements ultimately result in a net increase in activating signals in T cells and inhibitory signals in NK cells.

We demonstrate here that CD81 engagement leads to a global inhibition of NK cell activation, which extends beyond that exerted by KIR receptors. In particular, engagement of the HCV receptor CD81 by specific Ab or by the HCV glycoprotein E2 blocks NK cell activation induced by a wide range of stimuli, including fluid-phase stimuli such as IL-12, which are not inhibited by KIR-mediated, ITIM-dependent mechanisms 47. This is an important finding, since NK cells represent the main immune effector cells in early antiviral responses through their rapid and potent cytotoxic activity and their potential to produce inflammatory and antiviral cytokines.

The ability to modulate NK and T cell activity is not limited to CD81, but can be extended to CD82. The fact that different tetraspanins mediate the same effects may be due to their reported tendency to associate with partner molecules and with other tetraspanins to form an extended network of proteins. Here, CD81 and CD82 may interact either among each other or with the same partner proteins to mediate the same functions.

We also investigated the signaling events involved in the opposite NK and T cell responses to tetraspanin engagement. We show here that in T cells stimulated by suboptimal TCR-signals, the phosphorylation of Erk is increased by antibody- or HCV-E2-mediated engagement of CD81. In contrast, in NK cells, Erk phosphorylation is decreased by CD81-engagement 29. This is in line with previous results showing that overall tyrosine-phosphorylation and phosphorylation of the zeta chain, the most upstream event in TCR- and CD16-mediated signaling, are enhanced in T cells and reduced in NK cells by tetraspanin engagement 24, 26, 29, 40.

Our biochemical findings fully reflect the opposite functional effects of CD81 ligation on T and NK cells. One possible explanation for this dichotomy is that inhibitory (e.g. phosphatases) or stimulatory (e.g. kinases) molecules associate differently with CD81 upon engagement in the two cell types. For instance, inhibitory receptors of the KIR family are important negative regulators of NK cell activation but largely absent in T cells. A number of co-immunoprecipitation experiments did not reveal a direct association of CD81 with KIR receptors or any of the src homology 2 domain-bearing tyrosine phosphatases (SHP1, SHP2) that associate with them in NK cells. However, since IL-12 mediated NK cell activation is not inhibited by KIR receptor engagement 47 while IL-12 signaling is also inhibited by CD81 engagement, it is unlikely that tetraspanins act through a KIR-related mechanism.

Our data indicate that CD81 ligation enhances or reduces both proximal and distal signals induced by CD3 or CD16 and therefore appears to act on the general signaling efficiency, rather than inducing an independent pathway that converges further downstream with those dependent on CD3 or CD16. Therefore, we investigated raft microdomains or the cytoskeleton as possible intervention points. Here we demonstrate that CD81 is equally present in the raft fractions of both T and NK cells. The fact that forced raft association had the same co-stimulatory function on both T and NK cells indicates that the functional outcome of CD81 engagement is not merely due to ligation-induced raft clustering.

CD81 ligation alone did not induce any detectable biochemical or functional effects with the exclusion of morphological changes in both T and NK cells. Therefore, cytoskeleton rearrangement may be a central effect of CD81 engagement, as already demonstrated for other tetraspanins. In particular, engagement of CD82 on Jurkat cells was shown to induce morphological changes that are dependent on Rho-GTPase activity 26. We found that CD81 engagement enhances F-actin capping of both T and NK cells, which is consistent with a recent study on a number of tetraspanins on Jurkat cells 42. We also show that unrelated NK cell activation pathways, such as those triggered by CD16 engagement or by IL-12, are blocked by tetraspanins and activated by F-actin blockers, whereas T cell stimulation through CD3 is reduced by low doses of three different actin blockers.

Valitutti et al. 36 have previously shown that in a T cell-APC based system, cytoskeleton requirement for activation is high, whilst the use of soluble anti-CD3 mAb cross-linked by soluble goat-anti-mouse Ab activates T cells in a cytoskeleton-independent manner and is not blocked by CCD. We confirm these findings and show that activation by plastic-immobilized mAb is comparable to that in a two-cell system. As for NK cells, activation by both solid-phase and fluid-phase anti-CD16 mAb is greatly enhanced by actin blockers, indicating that cytoskeleton-mediated effects on NK cells extend to both types of stimuli. This is in line with the observed CCD-mediated enhancement of IL-12 induced cytokine production by NK cells and suggests fundamental differences in the way the cytoskeleton modulates T and NK cell activation. In addition, these findings explain why CD81 engagement can affect IL-2- and IL-12-dependent stimulation of NK cells 29, 30 while it has no effect on IL-2 stimulation of T cells 22.

The surprising finding that actin blockers enhance NK cell activation suggests that the reorganization of the cytoskeleton, while helping to maintain or propagate the signal in T cells, may contribute to its extinction in NK cells, possibly through the recruitment of molecules that participate in negative signaling in NK cells. While actin blockers inhibit these effects, tetraspanin engagement facilitates cytoskeleton rearrangement and thereby may influence the general ability of a cell to amplify or extinguish a signal. This model is supported by the finding that CD81-dependent NK cell inhibition is reduced and eventually abolished by CCD in a dose-dependent manner.

It is however clear from a number of publications that the cytoskeleton plays important positive roles in NK cell migration, contact with target cells and polarization for directed release of lytic granule content, indicating that our observations are only one aspect of cytoskeleton function in NK cells. A recently published example for cytoskeleton involvement in negative signaling is the actin-dependence of KIR receptor recruitment to the inhibitory synapse 48. We favor, however, a mechanism that is KIR independent, as cytokine-induced NK cell activation is blocked by the engagement of tetraspanins (as shown here) but not of KIR 47.

A striking parallel to the situation described here is found in mast cells, the only other cell type beside NK cells that is inhibited by CD81 engagement 27. In fact, pretreatment of mast cells with CCD or Latrunculin B increases activation of these cells, just as it does in NK cells 4951. Since the activity of both cell types is tightly controlled by negative signaling, and activation of both cell types is blocked by CD81 engagement and enhanced by actin blockers, we speculate that similar mechanisms may be in action.

We propose that tetraspanins, through their effect on cytoskeleton rearrangement, can modulate activation thresholds in a differential manner depending on the cell type. This explains the dichotomy of tetraspanin action, leading to enhanced activation of T cells and reduced activity of NK cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

Antibodies and reagents

Antibodies used were: anti-CD3 (clones OKT-3 and TR-66, ATCC, Rockville, MD), anti-CD16 (3G8, ATCC), anti-CD56 (B159.5, kindly provided by Dr. G. Trinchieri, Lyon, France), anti-CD81 (JS-81, PharMingen, San Diego, CA), anti-HLA class I (W6/32, Serotec, Oxford, GB), anti-CD82 (50F11, PharMingen), anti-β1integrin (M-106 Santa Cruz Biotechnology), anti-NKG2D (R&D Systems, Minneapolis, MN), rabbit anti-cholera toxin serum (Sigma, St. Louis, MO), goat-anti mouse-IgG F(ab)2 fragments (Sigma), anti-HCV E2 52. The anti-IFN-γ (B133.1 and B133.5), anti-TNF-α (B145.9 and B154.7) mAb were generously provided by Dr. G. Trinchieri.

The anti-p42/p44 Erk-2, anti-phospho p42/p44 Erk-2 (Cell Signaling Technology, Beverley, MA) and anti-LAT (2E9 Upstate Biotechnology) were used for Western blotting. Other reagents used were human recombinant IL-12 (R&D Systems), Cholera toxin B subunit (Sigma), CCD, Latrunculin A, Latrunculin B (Calbiochem, San Diego, CA) and recombinant purified HCV E2 53.

Cell preparation and cultures

PBMC were prepared from peripheral blood by Ficoll-Paque density gradient centrifugation, followed by a 1-h incubation in plastic flasks to remove adherent monocytes. NK cell cultures were prepared as described 29. Briefly, PBMC were cultured in 24-well plates at 5 × 105 cells/mL in RPMI-1640 medium supplemented with 10% FCS at 37°C together with the EBV-transformed B cell line RPMI-8866 irradiated at 50-Gy (5 × 104 cells/mL). On day 8 of the culture, cells were collected, and NK cells (>98% CD56+/CD3/CD19/CD14) were purified by depletion of the magnetically labeled CD3+/CD14+/CD19+ cells using MACS Separation Columns (Miltenyi Biotech, Gladbach, Germany).

Primary T cell lines were expanded from fresh PBMC by adding 1 μg/mL PHA to the culture, were maintained in 100 U/mL IL-2 from culture day 3 onwards and used for experiments at culture day 14.

Antibody coating and cell stimulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

The following purified mAb were used in stimulation experiments: anti-CD16 (3G8), anti-CD3 (TR-66), anti CD81 (JS-81), anti E2 (291A2), anti-CD56 (B159.5), anti-HLA class I (W6/32), anti-CD82 (50F11), anti-β1integrin (M-106).

Unless otherwise indicated, stimulation experiments were performed using plastic-immobilized mAb. The 96-well plates were coated as previously described 22. For coating the recombinant purified HCV E2 protein 53 onto the anti-E2 mAb (10 μg/mL), plates were washed with PBS after saturation and incubated with E2 (10 μg/mL in PBS) for 60 min at 37°C. After washing plates with PBS, cells were added in complete medium. In some experiments, cells were preincubated for 30 min at 37°C in the presence of CCD, Latrunculin A or Latrunculin B prior to stimulation.

Cytokine production assays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

Cells were cultured in 96-well plates for 24 h and the supernatants were assessed by ELISA for IFN-γ and TNF-α. The capture antibodies were anti-IFN-γ mAb B133.1 and the anti-TNF-α mAb B154.9, respectively, which were immobilized on ELISA plates. Detection of each cytokine was achieved using biotinylated anti-IFN-γ mAb B133.5 and anti-TNF-α mAb B154.7, streptavidin-conjugated Peroxidase (Sigma) and the chromogenic substrate o-phenylenediamine dihydrochloride (Sigma). Spectrophotometric analysis was performed at 450 nm on a Spectramax 340 spectrophotometer using Softmax Pro software (Molecular Devices, Sunnyvale, CA). Results are average of duplicate or triplicate cultures.

Immunoblot analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

Cells (106) were incubated on ice for 15 min with anti-CD16 mAb (3G8), anti-CD81mAb (JS-81, 10 μg/mL) or anti-CD3 mAb (TR-66). Cells were then washed and incubated with goat anti-mouse IgG F(ab)2 fragments (Sigma) at 30 μg/mL at 37°C for the indicated times. Following stimulation, cells were washed and lysed in buffer containing 20 mM Tris-HCl pH 7.4, 40 mM NaCl, 5 mM EDTA, 1 mM NaF, 20 mM Na4P207, 1 mM Na3VO4, 0.1% BSA, 1 mM PMSF, 5 μg/mL aprotinin, 10 μg/mL leupeptin, and 1% Triton X-100. Insoluble material was removed by centrifugation for 15 min at 15 000 × g. Cell extracts were resolved by SDS-PAGE and transferred to nitrocellulose membranes.

The p42/p44 and phospho p42/p44 MAP kinases were detected using Cell Signaling Technology antibodies, followed by donkey anti-rabbit IgG coupled to horseradish peroxidase (Amersham).

Isolation of lipid rafts

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

Lipid rafts were isolated by lysis in 1% Triton X-100 and floatation on sucrose density gradients as previously described 54. Briefly, 108 cells were incubated on ice for 30 min in 1 mL of 1% Triton X-100 in TNE (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) with protease and phosphatase inhibitors. Lysates were then centrifuged at 900 × g for 10 min to remove nuclei and cellular debris. Cleared supernatants were diluted 1:1 with 1 mL of 85% sucrose in TNE and layered at the bottom of a Beckman 14 × 89-mm centrifuge tube. The lysates were overlaid with 6 mL of 35% sucrose in TNE and 3.5 mL sucrose in TNE. Gradients were centrifuged at 200 000 × g in a SW40 rotor for 20 h at 4°C. At the end of the run, 1-mL fractions were collected from the top of the gradient, proteins were concentrated by 15% TCA precipitation and resolved by SDS-PAGE. Detection of CD81 and LAT was achieved by Western blotting with specific mAb (anti-CD81, JS-81; anti-LAT, 2E9).

Cell morphology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

Cells (2 × 105) were incubated on plates coated as described above, incubated in complete medium for 4 h at 37°C and photographed on a Zeiss Axiovert 25 light microscope (Zeiss, Oberkochen, Germany) at a magnification of 100x.

F-actin capping experiments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

Cells were incubated with 10 μg/mL of the indicated mAb for 20 min at 4°C. After washing in PBS, capping was induced by incubation for 1 h at 37°C on 96-well plates coated with 30 μg/mL of goat anti-mouse Ab. Cells were then collected, washed twice in PBS 1% FCS, and fixed by incubation at 106 cells/mL for 10 min at 4°C in IC Fix™ (Biosource International, USA). After two washes in PBS 1% FCS and two in ICPerm™, cells were resuspended in 40 μL of ICPerm™ with 0.77 µM TRITC-labeled phalloidin (Sigma-Aldrich, Milan, Italy) and incubated in the dark for 40 min at room temperature. Cells were then washed with PBS 1% FCS and confocal microscopy was carried out on a Leica Microsystems confocal microscope (Heidelberg, Germany).

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Note added in proof

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Antibody coating and cell stimulation
  8. Cytokine production assays
  9. Immunoblot analysis
  10. Isolation of lipid rafts
  11. Cell morphology
  12. F-actin capping experiments
  13. Note added in proof

The manuscript referred to in the text as ‘submitted for publication’, which describes the consequences of the appropriate engagement of CD81 alone on B cells, has now been accepted for publication. The publication details are:

Rosa, D., Saletti, G., De Gregorio, E., Zorat, F., Comar, C., D'Oro, U., Nuti, S. et al., Activation of naive B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc. Natl. Acad. Sci. USA 2005. 102: 18544–18549.