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Journal of Virology, January 2007, p. 474-482, Vol. 81, No. 2
0022-538X/07/$08.00+0     doi:10.1128/JVI.01777-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Switch from Latent to Productive Infection in Epstein-Barr Virus-Infected B Cells Is Associated with Sensitization to NK Cell Killing

Isabel Y. Pappworth,¹ Eddie C. Wang,¹ and Martin Rowe^1,2*

Department of Medical Biochemistry and Immunology, Wales College of Medicine, Cardiff University, Heath Park CF14 4XX, United Kingdom,¹ Division of Cancer Studies, University of Birmingham Medical School, Edgbaston, Birmingham B15 2TT, United Kingdom²

Received 16 August 2006/ Accepted 25 October 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Following activation of Epstein-Barr virus (EBV)-infected B cells from latent to productive (lytic) infection, there is a concomitant reduction in the level of cell surface major histocompatibility complex (MHC) class I molecules and an impaired antigen-presenting function that may facilitate evasion from EBV-specific CD8⁺ cytotoxic T cells. In some other herpesviruses studied, most notably human cytomegalovirus (HCMV), evasion of virus-specific CD8⁺ effector responses via downregulation of surface MHC class I molecules is supplemented with specific mechanisms for evading NK cells. We now report that EBV differs from HCMV in this respect. While latently infected EBV-positive B cells were resistant to lysis by two NK lines and by primary polyclonal NK cells from peripheral blood, these effectors efficiently killed cells activated into the lytic cycle. Susceptibility to NK lysis coincided not only with downregulation of HLA-A, -B, and -C molecules that bind to the KIR family of inhibitory receptors on NK cells but also with downregulation of HLA-E molecules binding the CD94/NKG2A inhibitory receptors. Conversely, ULBP-1 and CD112, ligands for the NK cell-activating receptors NKG2D and DNAM-1, respectively, were elevated. Susceptibility of the virus-producing target cells to NK cell lysis was partially reversed by blocking ULBP-1 or CD112 with specific antibodies. These results highlight a fundamental difference between EBV and HCMV with regards to evasion of innate immunity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epstein-Barr virus (EBV) is a member of the gammaherpesvirus family and is found in more than 90% of the adult population worldwide. Following primary infection, it persists in the B-lymphocyte population. In a healthy host, the majority of infected B cells exist in a resting phenotype with limited gene expression (31, 39, 51, 52). Expression of a further 10 latent genes can lead to growth transformation of B cells (22, 48, 51), which is thought to be the main route for expansion of the EBV pool within the infected host. In addition to this and like alpha- and betaherpesviruses, periodic activation of the full lytic cycle with production of infectious virions aids expansion of the virus within the host and enables its transmission to new hosts.

The growth-transforming potential of EBV necessitates efficient host defense mechanisms to ensure that latently infected B cells do not proliferate unchecked. CD8⁺ T-cell responses to EBV-encoded proteins expressed in growth-transformed cells are important components of the host immune response that restrict the outgrowth of EBV-transformed B cells (19, 45).

T-cell responses are also mounted to a number of lytic cycle genes, but the efficiency of these responses appears to be limited by immune evasion mechanisms. Thus, in early lytic cycle, EBV reduces expression of HLA class I molecules on the infected cell surface (23) and interferes with the ability of the transporters associated with antigen processing (TAP-1 and TAP-2) to translocate immunogenic peptides from the cytosol to the endoplasmic reticulum where they would associate with HLA class I molecules (40). EBV encodes at least two early lytic cycle genes that have been shown to block the synthesis of new HLA class I molecules and to interfere with the function of TAP (M. Rowe and A. Hislop, unpublished data). In addition, the late gene product viral interleukin 10 (IL-10) has been reported to downregulate expression of TAP-1 and, consequently, the level of cell surface HLA class I molecules (66). There is evidence to suggest that these effects on the HLA class I antigen-processing pathway do impact on the CD8⁺ immune response in vivo (38). EBV also directly targets the HLA class II pathway in the lytic cycle through expression of a late membrane glycoprotein, gp42, that binds to HLA class II molecules to cause steric hindrance of T-cell recognition (41, 42). The immediate-early gene BZLF1 inhibits gamma interferon (IFN-) receptor expression, thus preventing activation of IFN- signaling pathways whose targets include induction of HLA class I and class II molecule expression; BZLF1 expression might therefore act to impair T-cell responses to EBV (34).

In common with other herpesviruses (55, 57, 64), therefore, EBV has multiple mechanisms to increase the likelihood of evading adaptive T-cell responses to the large number of virally encoded proteins expressed in the lytic cycle. However, a reduction in MHC class I molecule expression on the cell surface risks exposure to the innate immune system, specifically to NK cell attack. Acting via multiple activating and inhibitory receptors with a diverse range of structures and signaling properties, NK cells play an important role in host defense against virally infected cells (7, 11, 15). There is accumulating evidence that many viruses that cause persistent infections, including herpesviruses, have evolved mechanisms to evade NK cell responses and T-cell responses (27, 36).

Cytomegaloviruses, members of the betaherpesvirus family, represent a paradigm for NK cell evasion. For human cytomegalovirus (HCMV), the UL18 glycoprotein has homology to class I major histocompatibility complex (MHC) heavy chains (5) and appears to imitate class I MHC molecules engaging inhibitory NK cell receptors (13, 43). A second MHC class I homologue, UL142, has likewise been shown to confer protection against NK cells (61). UL16 sequesters the ligands MicB and ULBP-1 to ULBP-3 that otherwise interact with the activating NK receptor NKG2D (59). The nonclassical MHC class I molecule, HLA-E, has been shown to interact specifically with C-type lectin receptor CD94/NKG2A/B on NK cells to deliver an inhibitory signal. HLA-E exhibits limited polymorphism and binds a restricted set of peptides derived from the leader sequence of classical MHC class I molecules and HLA-G, via a TAP-dependent pathway (10, 26). This source of HLA-E-binding peptides is blocked by HCMV as part of the strategy for evasion of CD8⁺ T cells, but UL40 delivers an HLA-E-binding peptide in a TAP-independent manner to the endoplasmic reticulum, thus upregulating surface HLA-E expression and inhibiting CD94/NKG2A⁺ NK cells (53, 58). Finally, UL141 downregulates surface CD115, a ligand for the NK-activating receptor CD226 (54).

While the evidence for NK cell evasion by HCMV is overwhelming, it is not known whether EBV has equivalent NK cell evasion mechanisms. B cells of EBV-transformed lymphoblastoid cell lines (LCLs) displaying latent infection are generally resistant to NK cell lysis, although lines such as LCL 721.221 with genetic aberrations resulting in loss of cell surface HLA class I expression or normal LCLs incubated with blocking immunoglobulin M (IgM) antibodies to HLA class I molecules are sensitive to NK cells and are often used as positive control targets in NK cell studies (37, 47, 60). The effect of EBV lytic cycle induction on the sensitivity to NK cells has been previously investigated, and the results were interpreted as showing increased sensitivity to NK cell killing (8). In these experiments, the lytic cycle was induced with sodium butyrate, which activates a broad spectrum of host genes independently of induction of cells into the lytic cycle, or by superinfection with a laboratory mutant EBV. Crucially, in neither case was it possible to ascertain whether the induction of NK lysis was specific for the subpopulation of cells in the lytic cycle.

A knowledge of the NK cell sensitivity of EBV-infected cells is essential to understand the complex and finely balanced interactions between EBV and the immune response, both in persistently infected healthy individuals and in EBV-associated diseases. We recently developed a model system for isolating and studying EBV-infected B cells in lytic cycle (40). This system was used in the present study to reexamine the question of whether the repertoire of EBV lytic cycle genes includes NK cell evasion functions. In contrast to what is observed with HCMV, EBV lytic cycle in B cells was associated with a pronounced increase in sensitivity to NK cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells. AKBM cells, derived from the EBV-positive Japanese Burkitt's lymphoma cell line Akata by stable transfection of the pHEBO-BMRF1p-rCD2/GFP reporter plasmid and selection with 0.5 mg/ml hygromycin B, have been described previously (40). AK31, an EBV-loss subclone of Akata, was transfected with pEGFPN1-rCD2, and a stable transfectant was selected by growth in 1 mg/ml G418 (40). K562 is a proerythromyeloleukemic cell line (29) that expresses low levels of HLA class I molecules and is sensitive to lysis by NK cells. The above-mentioned lines were propagated as suspension cultures in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 200 U/ml penicillin, and 200 mg/ml streptomycin (RPMI-FCS).

DEL NK cells, derived from a patient with lymphoid dysplasia associated with peripheral demyelinating neuropathy (58), were propagated as suspension cultures in RPMI 1640 medium with 10% AB serum, 2 mM L-glutamine, 200 U/ml penicillin, and 200 mg/ml streptomycin (RPMI-AB) supplemented with 500 IU/ml IL-2. The NK-L cell line, derived from a patient with aggressive natural killer cell leukemia, was maintained in RPMI-FCS supplemented with IL-2, which was reduced prior to use in killing assays as described previously (44). Primary NK cell bulk cultures were generated by depletion of adherent cells and T cells from peripheral blood mononuclear cells from healthy adult donors using a monoclonal antibody (MAb) to CD3 (Serotec, Oxford, United Kingdom) and anti-mouse IgG Dynabeads (Dynal, Bromborough, United Kingdom) as instructed by Dynal. The NK-enriched cells were then incubated overnight in RPMI-AB and 1,000 IU/ml IFN- before use. Allospecific T cells were activated and expanded by cocultivation of peripheral blood mononuclear cells from a healthy adult donor with irradiated Akata cells in supplemented RPMI-AB with IL-2. The T cells were challenged with irradiated Akata cells at a responder:stimulator ratio of 4:1 on days 1, 7, 14, and 22, and 20 IU/ml IL-2 was added on days 7, 11, 14, 18, and 22.

Antibodies. BZ.1 is a murine MAb to the Zta immediate-early protein encoded by the EBV BZLF1 gene (65). OX34, a MAb specific for rat CD2, and W6/32, a MAb specific for MHC class I molecules (HLA-A, -B, and -C alleles), were produced from hybridomas purchased from the American Tissue Culture Collection. The MAb specific for CD112 and red phycoerythrin (RPE)-conjugated MAbs specific for mouse IgG HLA-DR, W6/32, and CD80 were purchased from Serotec, Oxford, United Kingdom. MAbs specific for ULBP-1, MicA, and MicB, the recombinant human NKG2D/Fc chimera, and the secondary Alexa Fluor 647-conjugated anti-human Fc were purchased from R&D Systems, Europe. MAbs specific for CD155 (PVR), nectin, and HLA-E (clone MEM/E/08) were purchased from Exbio, Prague, Czech Republic. Unconjugated murine MAbs specific for human CD2, CD11b, CD22, CD29, CD31, CD34, CD36, CD41, CD42b, CD43, CD44, CD48, CD49e, CD50, CD54, CD56, CD58, CD147, and CD162 were purchased from Immunotools, Germany. Alexa Fluor 647-conjugated (Molecular Probes, Europe) or allophycocyanin-conjugated (Caltag Laboratories) goat anti-mouse IgG were used as secondary antibodies. RPE-conjugated anti-mouse IgG1 subclass secondary antibodies were purchased from Serotec, Oxford, United Kingdom.

Induction of EBV lytic cycle. AKBM cells (3 x 10⁷) were washed out of culture media and resuspended in 4 ml RPMI-FCS with 1% goat anti-human IgG (Cappel, OH) and incubated at 37°C for 2 h. Cells were then washed in phosphate-buffered saline (PBS) and returned to a flask with fresh RPMI-FCS at 10⁶ cells/ml until required for use at 6 to 24 h postinduction.

Flow cytometry. For flow cytometry detection of BZLF1, cells were fixed and permeabilized to allow for detection of the nuclear protein. Cells were fixed for 30 min at 0°C with 2% paraformaldehyde solution in PBS and then permeabilized by the addition of an equal volume of 0.4% Triton X-100 in paraformaldehyde solution and incubated for a further 30 min at 0°C. After the cells were washed with PBS, they were incubated for 1 h at 37°C with MAb BZ.1 (anti-BZLF1) at 0.5 µg/ml in PBS with 10% normal rabbit serum. Cells were washed twice with PBS and then incubated with 1 µg/ml RPE-conjugated goat anti-mouse IgG1 for 1 h at 37°C. After further washes with PBS, the cells were resuspended in 2% paraformaldehyde in PBS and stored at 4°C in the dark until analysis. For cell surface antigens, viable cells were washed out of medium and kept at 0 to 4°C to minimize cell activity. The cells were incubated for 1 h at 0°C with an appropriate dilution of primary antibody in PBS and 10% normal rabbit serum. Cells were washed and incubated with 0.2 µg/ml Alexa Fluor 647- or allophycocyanin-conjugated goat anti-mouse IgG for 1 h at 0°C. After further washes, the cells were resuspended in 2% paraformaldehyde in PBS and analyzed on a FACSCalibur flow cytometer (Becton Dickinson Co., San Jose, CA). Flow cytometers were provided by the Flow Facility of the Central Biotechnology Service, Wales College of Medicine, Cardiff University, Wales, United Kingdom.

Immunomagnetic separation. At 24 h following induction of lytic cycle with anti-human IgG antibodies, viable AKBM cells expressing the rat CD2/green fluorescent protein (GFP) reporter were stained with MAb OX34 and then positively selected by using magnetic cell sorting with anti-IgG2a plus IgG2b microbeads and MS columns (Miltenyi Biotech) as described previously (40). A purity of 85 to 95% GFP-positive cells was routinely achieved.

Cytotoxicity assay. Chromium release cytotoxicity assays were performed as previously described (33). In some experiments, antibody blocking assays were performed by preincubating the ⁵¹Cr-loaded target cells for 30 min with blocking antibody at the indicated concentrations before the addition of effector cells.

Cell adhesion assay. Target cells were washed out of media and incubated for 15 min at 37°C in 10 mM carboxy-fluorescein diacetate succinimidyl ester (Sigma, Poole, United Kingdom). NK cells were incubated similarly with Cell Trace Far Red (Molecular Probes, Europe). Excess dye was washed out with RPMI-FCS before mixing effector and target cells at a 1:1 ratio. Cells were brought into proximity by centrifugation for 3 min at 18 x g before incubation at 37°C for 40 min. Cells were fixed in 2% paraformaldehyde in PBS before flow cytometric analysis for conjugated cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of Akata target cells in the lytic cycle with reduced MHC class I expression. We previously reported the generation and characterization of the AKBM cell line that was derived from the EBV-positive Burkitt's lymphoma cell line Akata by transfection with a reporter to express an immunosortable surface marker upon induction of the lytic cycle (40). Ligation of the B-cell receptor (BCR) with anti-human IgG antibody typically led to 15 to 45% of the AKBM cells entering the lytic cycle as shown by intracellular immunofluorescence staining with the BZ.1 MAb recognizing the immediate-early protein Zta (BZLF1). Coincident with lytic cycle induction, the chimeric reporter gene rat CD2/GFP was expressed at the cell surface almost exclusively on those cells costaining for Zta (Fig. 1A, left panel). Staining of viable cells with W6/32 antibody demonstrated reduced expression of HLA class I molecules on the surfaces of those cells expressing the rat CD2/GFP reporter (Fig. 1A). Following expression of Zta at 2 to 4 h postinduction (data not shown), a reduction of HLA expression is first observed at around 6 h, and a maximum reduction was observed by 12 to 24 h (Fig. 1B). The expression of the rat CD2/GFP reporter allows a rapid and efficient isolation of cells in the lytic cycle by immunomagnetic sorting (Fig. 1C).

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FIG. 1. Isolation of AKBM cells in the lytic cycle and reduced expression of cell surface HLA class I molecules. (A) Fluorescence analysis of AKBM cells at 24 h postligation of the BCR. Entry into the lytic cycle is indicated by expression of the rat CD2/GFP fusion protein reporter expressed from the early BMRF1 promoter and by intracellular staining with MAb BZ.1 to the immediate-early protein Zta. Viable cells stained with MAb W6/32 for surface HLA-A, -B and -C molecules demonstrate a reduction in HLA class I molecules on those cells expressing GFP. (B) Kinetics of the reduction in cell surface HLA class I molecules in the lytic cycle. The fluorescence intensity of W6/32 MAb staining on viable AKBM cells was measured at several time points up to 24 h after stimulation into the lytic cycle. The results were calculated as a ratio of the mean fluorescence intensity of W6/32 staining on GFP-positive cells to the mean fluorescence intensity of W6/32 staining on GFP-negative cells at each time point. The data are the means ± standard errors of the means (error bars) for three replicate induced cultures. (C) Histograms showing the efficiency of immunomagnetic sorting of induced AKBM cells using OX34 antibodies to rat CD2. The histograms show GFP expression in uninduced control AKBM cells (top panel), unsorted induced AKBM cells (middle panel), and positively sorted induced AKBM cells (bottom panel) in one representative experiment.

HLA-A, -B, and -C class I molecules are ligands for the KIR inhibitory receptor on NK cells (46). Therefore, unless EBV has NK cell evasion mechanisms similar to those of HCMV, the reduced expression of HLA-A, -B, and -C molecules potentially tips the balance in favor of EBV-infected cells in the lytic cycle being at risk to lysis by NK cells. We investigated this possibility by using the induced and sorted AKBM cells as targets in chromium release cytotoxicity assays with different NK effector cell populations.

Akata cells in the lytic cycle are more susceptible to NK killing. AKBM cells were induced into the lytic cycle with anti-IgG. After 24 h, the rat CD2/GFP-expressing cells were isolated by OX34 staining and magnetic bead separation as in Fig. 1C, then loaded with ⁵¹Cr, and used as targets in chromium release assays with various NK cell effectors. Figure 2A illustrates the results obtained in one representative experiment using the well-characterized NK cell line, NK-L, which has a CD94⁺ CD16⁺ CD56^dim NKG2A⁺ surface phenotype. Six different target cell lines were analyzed over a range of effector:target ratios from 40:1 to 2.5:1. Both the standard NK-sensitive target line K562 and the lytic cycle-induced AKBM targets were efficiently killed by NK-L effector cells in a dose-dependent manner, whereas there was no killing of uninduced AKBM cells even at the highest (40:1) effector:target ratio used. Additional controls were included to eliminate the possibility of an effect due to the antibodies used in the induction and sort. AK31, an EBV-negative subclone of the parental EBV-positive Akata line, was stably transfected with a constitutive CMV promoter-driven rat CD2/GFP vector. The resulting line, AK31-CD2/GFP, was subjected to the same anti-IgG BCR ligation and OX34 magnetic beads isolation procedure as the AKBM cells and was then included as a target in the same chromium release assays. As shown in Fig. 2A, there was no significant specific lysis of the induced and sorted AK31-CD2/GFP targets at any of the effector:target ratios studied. Likewise, there was no specific lysis of either the uninduced AK31-CD2/GFP cells or the parental AK31 line.

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FIG. 2. Sensitivity of lytic cycle B cells to NK lysis. Chromium release cytotoxicity assays were performed to determine the percentage of specific lysis of the indicated targets following incubation for 4 h with NK cells at a range of effector:target ratios (40:1, 20:1, 10:1, 5:1, and 2.5:1). The NK cell effectors were NK-L cells (A), DEL NK cells (B), or primary NK cells from peripheral blood activated with IFN- (C). The data are the means plus standard errors of the means (error bars) for four replicate assays from one representative experiment with each type of effector cell.

Similar results were obtained with a second NK cell line, DEL, which displayed a CD94⁺ CD16⁺ CD56^bright NKG2A⁺ surface phenotype. As shown in the representative assay in Fig. 2B, the lytic cycle-induced AKBM targets were again killed at an efficiency similar to that of the standard NK cell sensitive K562 target line, whereas the latent AKBM targets were largely resistant to lysis by DEL effectors. The low level of specific lysis of latent AKBM cells at the higher effector:target ratios was reproducibly observed in replicate experiments.

The results with the two NK cell lines were confirmed using primary NK cells obtained from fresh peripheral blood mononuclear cells by T-cell depletion and overnight stimulation with IFN-. As before, the EBV-positive AKBM cells induced into the lytic cycle were markedly more susceptible to NK cell-mediated killing than were uninduced AKBM cells (Fig. 2C). This result was reproduced with primary NK cell preparations from all seven healthy adult donors (data not shown).

Although we have previously demonstrated that the cells induced into the lytic cycle do not show reduced viability within the first 3 or 4 days of the lytic cycle (40), we wanted to discount the possibility that the sensitivity of the cells to NK cell-mediated lysis was an artifact of the cells breaking down under the stress of the ⁵¹Cr release cytotoxicity assay. To address this, allospecific T cells were raised to latent Akata cells and were used as effectors in ⁵¹Cr release cytotoxicity assays with uninduced and sorted induced AKBM target cells. As expected, the uninduced AKBM cells were specifically killed in a dose-responsive manner over a range of effector:target ratios (Fig. 3). The induced AKBM cells, however, showed no significant lysis at effector:target ratios up to 20:1 and only a low level of lysis at 40:1.

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FIG. 3. Sensitivity of latent and lytic cycle AKBM cells to allospecific cytotoxic T lymphocytes. A chromium release cytotoxicity assay was performed with an allospecific effector T-cell line generated by stimulation of HLA-mismatched T cells with latent AKBM cells and IL-2. The target cells were uninduced latently infected AKBM cells (), lytic cycle sorted AKBM cells (•), and K562 cells (). The line graph indicates the means ± standard errors of the means (error bars) of the percentages of specific lysis calculated from four replicate samples in one representative experiment.

Together, the data in Fig. 2 and 3 demonstrate that induction of the virus productive cycle in AKBM cells was associated with a specific acquired sensitivity to lysis by NK cell effectors.

Adherence of AKBM cells to NK cells. As a first step to investigating the mechanism of this increased sensitivity of cells in the EBV lytic cycle to NK cell-mediated killing, we analyzed the expression of adhesion molecules on the target cells that might promote stronger or more prolonged contact between target and effectors. A panel of monoclonal antibodies to adhesion molecules was used to identify a possible candidate. Latent AKBM cells expressed low but significant levels of CD2, CD11b, and CD22 (Fig. 4A) and CD29, CD56, and CD58 (data not shown) and higher levels of CD43, CD48, and CD147 (Fig. 4A) at the cell surface. No detectable expression of CD31, CD34, CD36, CD41, CD42b, CD44, CD49e, CD50, CD54, or CD162 was detected on AKBM cells (data not shown). Expression of none of these antigens was increased during the lytic cycle. However, expression of both CD2 and CD11b was markedly reduced in AKBM cells that had been induced into the lytic cycle (Fig. 4A).

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FIG. 4. Adhesion of lytic cycle B cells and NK cells. (A) Flow cytometry histograms comparing the levels of adhesion molecules on AKBM cells in latent infection and in the lytic cycle. Staining obtained with the indicated monoclonal antibodies (CD2, CD11b, CD29, CD43, CD48, or CD147) is shown as unfilled line histograms, while background staining with a control antibody with an irrelevant specificity is indicated by the gray-shaded histograms. (B) Conjugate formation between NK-L cells labeled with Far Red fluorochrome and AKBM cells labeled with carboxy-fluorescein diacetate succinimidyl ester fluorochrome. The left-hand panel shows 13.4% conjugates between latent AKBM cells and NK-L cells. The right-hand panel shows 3.0% conjugates between lytic cycle AKBM cells and NK-L cells.

The adhesion molecule expression profile did not provide any evidence for the possibility of increased adhesion of induced AKBM target cells with NK cell effectors. To examine this more directly, conjugate formation assays were performed with NK-L cells. In the representative experiment shown in Fig. 4B, approximately 13% of the uninduced latent AKBM cells formed conjugates, but this was reduced to 3% of induced and sorted lytic cycle cells. Thus, we found no evidence for the increased susceptibility of lytic cycle-induced AKBM cells to NK cell-mediated killing being due to increased adhesion between these effectors and targets.

Altered expression of NK ligands on AKBM cells. We next examined whether expression of NK cell ligands other than HLA-A, -B, and -C molecules were altered following induction of AKBM cells into the virus productive cycle. Several NK cell-activating and -inhibitory receptors and their corresponding ligands have been reported in the literature. The balance of the ligands on the target cell for activating or inhibitory receptors on the NK cell effectors is an important factor determining sensitivity to NK cell-mediated lysis. The HLA-A, -B, and -C molecules recognized by MAb W6/32 are ligands for the KIR family of inhibitory receptors (46), so the observed reduction of these cell surface molecules (Fig. 1) is likely to favor increased NK cell-mediated killing of lytic cycle AKBM cells. HLA-E, a nonclassical HLA class I molecule, whose surface expression usually parallels that of the other class I alleles, is a ligand for the CD94/NKG2A inhibitory receptors (10). Using the HLA-E specific MAb MEM-E/08, we observed weak expression of this HLA class I molecule on latent AKBM cells and significant reduction of expression following activation of lytic cycle (Fig. 5). As expected, HLA-E was not detected on the surface of the K562 cell line (Fig. 5).

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FIG. 5. Expression of NK cell ligands on lytic cycle B cells. Flow cytometry histograms showing the expression of different NK ligands on AKBM cells in latent infection, AKBM cells in the lytic cycle, and K562 cells. Staining obtained with the MAbs to the indicated antigens (HLA-E, ULBP-1, or CD112) is shown as unfilled line histograms, while background staining with a control MAb with an irrelevant specificity is indicated by the gray-shaded histograms.

Some NK ligands examined, such as nectin-1 and CD155, were undetectable on either latent or lytic AKBM cells (data not shown). Others, such as CD48, a ligand for the 2B4-activating receptor (24), were found to be equally expressed on latent and lytic AKBM cells (Fig. 4B). Interestingly, while an NKG2D-Fc fusion protein showed no detectable binding to latent AKBM cells, induced AKBM cells in the lytic cycle reproducibly showed weak but significant binding of this reagent (data not shown). MicA, MicB, ULBP-1, ULBP-2, and ULBP-3 have all been shown to interact with the NKG2D receptor (4, 49). While ULBP-2, ULBP-3, MicA, and MicB were all undetectable on AKBM cells during either the latent or lytic cycle (data not shown), ULBP-1 was almost undetectable on latent AKBM cells, but the level was reproducibly increased following induction of lytic cycle (Fig. 5). Finally, CD112 (nectin-2), a ligand for the DNAM-1-activating receptor on NK cells (9), was also elevated on the surfaces of cells induced into the lytic cycle (Fig. 5).

The cell surface phenotype therefore suggested that expression of two activating ligands, ULBP-1 and CD112, was induced following entry of AKBM cells into the lytic cycle. The functional significance of elevated ULBP-1 and CD112 was examined by antibody blocking experiments. The inclusion of antibody in the cytotoxicity assays to block the ULBP-1 ligation caused a significant reduction in lysis of induced AKBM cells by NK-L and primary NK cell effectors (Fig. 6). Anti-CD112 antibodies showed a similar blocking of NK cell-mediated lysis of induced AKBM cells, although this did not reach statistical significance in every experiment (Fig. 6).

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FIG. 6. Blocking of NK lysis by ULBP-1 and CD112 MAbs. Sorted lytic cycle AKBM target cells were preincubated with 10 µg/ml irrelevant IgG control MAb, anti-ULBP-1 MAb, or anti-CD112 MAb for 30 min at 37°C prior to inclusion in a chromium release cytotoxicity assay with NK effectors at an effector:target ratio of 40:1. The percentages of specific lysis obtained in three independent experiments are shown. NK-L cells were used as effectors in experiment 1 (Exp 1), while primary NK cells from two different healthy donors were used as effectors in experiments 2 and 3. The significance of the effects of the ULBP-1 and CD112 MAbs relative to the control IgG in each experiment was determined using a t test assuming unequal variance as follows: P < 0.01 (***), P < 0.05 (**), and P = 0.06 (*).

Together, these data are consistent with AKBM cells in the virus productive cycle becoming sensitive to NK cell lysis through the combined effects of downregulated expression of HLA -A, -B, -C, and -E molecules, which are ligands for NK cell inhibitory receptors, and upregulated expression of ULBP-1 and CD112, which are ligands for NK cell-activating receptors.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The many mechanisms utilized by HCMV and other viruses to evade both T-cell responses (55, 57, 64) and NK cells (27, 36) has given interesting insight into the interactions of persistent human viruses with the host immune system. While the evasion of T-cell responses appears to be a common feature of all herpesviruses investigated so far, including EBV, the situation regarding evasion of NK cells is less clear. Our present data show that for EBV-infected B cells, the balance of inhibitory and activating NK ligands is altered upon induction of the virus lytic cycle, concomitant with an increase in sensitivity to NK cell-mediated lysis.

EBV has no obvious homologues of MHC class I molecules, such as the UL18 and UL142 proteins of HCMV, that might replace the KIR-binding functions of HLA-A, -B, and -C molecules to inhibit activation of NK cells (5, 61). Furthermore, EBV has no homologue of UL40, which in HCMV-infected cells provides a signal peptide sequence that binds HLA-E and maintains the expression of this nonclassical HLA class I molecule at the infected cell surface (58) to engage CD94/NKG2A inhibitory NK receptors (10). The levels of surface HLA class I molecules on the EBV-infected cells in the lytic cycle cannot be restored by treatment with IFN- or IFN- (M. Rowe, unpublished data), which is consistent with the BZLF1-mediated inhibition of IFN- signaling (34) and BGLF5-mediated repression of host de novo protein synthesis (M. Rowe, submitted for publication) that would negate any possible IFN-mediated transcriptional activation of HLA class I. EBV, therefore, appears to lack mechanisms for counteracting the downregulation of cell surface HLA class I molecules in the lytic cycle. In addition, we found increased expression of ULBP-1 and CD112, which are ligands for the activating NK cell receptors NKG2D (49) and DNAM-1 (9), respectively. The functional significance of these elevated activating ligands was confirmed in blocking antibody experiments (Fig. 6). Our data do not exclude the possibility that other as yet unidentified NK cell ligands might also be modulated. Taken together, our data point to the conclusion that EBV differs substantially from HCMV with regards to evasion of NK cells.

Following induction of EBV-infected B cells into the lytic cycle, we observed a marked increase in sensitivity to NK cell-mediated lysis by two clonal NK cell populations (NKL and DEL) as well as by polyclonal primary NK cell populations from all seven healthy adult donors tested. In common with other reports in the literature (37, 47), we find that normal EBV-transformed LCLs showing latent EBV infection are largely resistant to lysis by these effectors (data not shown). The high levels of HLA-A, -B, and -C molecules and of HLA-E molecules on the surfaces of LCLs are likely to be major factors conferring resistance to NK cell lysis, since mutant LCLs not expressing these surface molecules become sensitive targets (37, 60, 63). The general resistance of normal LCLs to NK cell-mediated lysis is sometimes overlooked when interpreting the role of NK cells in EBV-associated diseases, such as chronic active EBV infection (21, 30), X-linked lymphoproliferative disease (6, 37, 50), and posttransplant lymphoproliferative disease (56). In these patients, the contribution of NK cell defects to the life-threatening infectious mononucleosis and EBV-induced lymphoma is likely to involve a failure of the NK cells to control lytic EBV infection, with a resultant increase in the viral load and subsequently the frequency of freshly infected cells.

It has been reported that NK cells that do lyse latently infected LCLs can be activated from cord blood and from EBV-seronegative donors by cocultivation with autologous LCLs in vitro (32, 62). The nature of this NK cell response has not been fully characterized with regards to the NK cell receptors involved or to their ability to kill lytically infected cells. Nor is it clear why such responses are not seen in adult seropositive donors. A role for these NK cells during primary EBV infection was postulated (32, 62).

With regards to primary EBV infection, Williams et al. recently reported that NK cell numbers in blood are elevated in patients with acute infectious mononucleosis and although the NK cells from these patients killed an HLA-negative LCL, they did not kill autologous LCLs very efficiently (60). These authors also observed an inverse correlation between the percentage of NK cells in peripheral blood and the viral load in peripheral mononuclear cells at the time of diagnosis of acute infectious mononucleosis (60). While our results would indicate that the NK cells preferentially target EBV-infected B cells in the lytic cycle, the increased viral load in the peripheral mononuclear cells in infectious mononucleosis patients is mostly due to latently infected cells, since lytic gene products are only rarely detected in the blood of infectious mononucleosis patients or in healthy persistently infected individuals (14, 20). The effect of NK cells on the viral load in primary infection is likely, therefore, to be mediated predominantly in the secondary lymphoid tissues, such as tonsils, where lytic EBV infection can be observed (1, 2, 25, 35).

With regards to the role of NK cells in controlling persistent EBV infection, it is interesting to note that in healthy individuals, the percentage of NK cells in tonsils, typically between 0.2 and 1%, is around 50 to 100 times lower than the percentage of NK cells in the peripheral blood or spleen (17, 18, 28). Furthermore, while NK cells in the peripheral blood are mostly CD56^dim, express perforin, and are cytolytic, the NK cells in tonsils are predominantly CD56^bright, generally lack perforin expression, and are relatively noncytolytic. The tonsil NK cells can respond to T-cell-derived IL-2 to express both perforin and cytokines (12, 16, 18). In this context, it should be noted that our cytotoxicity assays on primary NK cells from peripheral blood samples were performed with NK cells that had been treated with IFN- to enhance the effector function, since untreated NK cell effectors show limited cytotoxicity against K562 targets at the effector:target ratios used. Interestingly, untreated NK cells demonstrated significant cytotoxicity of lytic AKBM cells in the same assays, and further experiments indicated that supernatants from lytic AKBM cells were able to activate the NK cells to kill K562 targets (data not shown). These results have implications for attempts to interpret the significance of the phenotypically different NK cell populations at different anatomical sites in relation to the control of EBV. Initiation of lytic cycle in B cells not only results in increased sensitivity to NK cell-mediated lysis but also leads to the production of a soluble factor(s) able to enhance NK cell function. We are currently investigating the nature of these factors and how different populations of NK cells might be affected.

Not withstanding the fact that phenotypically different NK cell populations are found at different anatomical sites, on the basis of the frequency of NK cells alone, it is possible that tonsils might normally be a relatively permissive environment for lytic EBV replication. In contrast, the abundance of potentially cytolytic NK cells in the periphery and possibly in other secondary lymphoid tissues would efficiently eliminate lytically infected B cells from these sites. Immunohistochemical studies on tonsils from patients with acute infectious mononucleosis suggest that very rare cells expressing lytic cycle products are B cells (1, 35), and sensitive PCR analysis of tonsillar subpopulations indicates that these cells have a plasma cell phenotype (25). The rarity with which lytically infected B cells can be detected in vivo hampers attempts to define where lytic cycle activation might normally occur. Furthermore, the rarity of detection could be a consequence of infrequent lytic activation or it might reflect the efficiency of NK cell-mediated control over these cells.

The colonization of the B-lymphocyte compartment by EBV (2, 31) might underlie the fundamental difference between EBV and HCMV with regards to the need to develop NK cell evasion mechanisms. EBV does not need to undergo lytic replication at sites where NK cells are abundant, but limited lytic replication in tonsils might be beneficial for new infections of naïve B-cell populations that subsequently traffic through the germinal centers to emerge as memory B cells showing a restricted form of latent infection (3). The relatively low frequency of NK cells in the tonsil might, therefore, serve to maintain the number of EBV-infected memory B cells in the peripheral blood.

In summary, we have demonstrated that EBV-infected B cells that enter the lytic cycle become sensitive to NK cell killing. Our observations reinforce the growing evidence implicating NK cells, together with EBV-specific cytotoxic T cells, as important effectors regulating the potential pathogenicity of EBV infection.

 
This study was funded by a project grant award (GR072425) from the Wellcome Trust, London, United Kingdom.

We thank Sian Llewellyn-Lacey of the MRC Tissue Culture Facility at the Wales College of Medicine and Wendy Thomas at Birmingham University for their technical support and Caroline Rowe for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Division of Cancer Studies, University of Birmingham Medical School, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 44 121 4147144. Fax: 44 121 4144486. E-mail: m.rowe{at}bham.ac.uk.

Published ahead of print on 1 November 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Anagnostopoulos, I., M. Hummel, C. Kreschel, and H. Stein. 1995. Morphology, immunophenotype, and distribution of latently and/or productively Epstein-Barr virus-infected cells in acute infectious mononucleosis: implications for the interindividual infection route of Epstein-Barr virus. Blood 85:744-750.[Abstract/Free Full Text]
2
2. Babcock, G. J., L. L. Decker, M. Volk, and D. A. Thorley-Lawson. 1998. EBV persistence in memory B cells in vivo. Immunity 9:395-404.[CrossRef][Medline]
3
3. Babcock, G. J., and D. A. Thorley-Lawson. 2000. Tonsillar memory B cells, latently infected with Epstein-Barr virus, express the restricted pattern of latent genes previously found only in Epstein-Barr virus-associated tumors. Proc. Natl. Acad. Sci. USA 97:12250-12255.[Abstract/Free Full Text]
4
4. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, and T. Spies. 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727-729.[Abstract/Free Full Text]
5
5. Beck, S., and B. G. Barrell. 1988. Human cytomegalovirus encodes a glycoprotein homologous to MHC class-I antigens. Nature 331:269-272.[CrossRef][Medline]
6
6. Benoit, L., X. Wang, H. F. Pabst, J. Dutz, and R. Tan. 2000. Defective NK cell activation in X-linked lymphoproliferative disease. J. Immunol. 165:3549-3553.[Abstract/Free Full Text]
7
7. Biron, C. A., K. S. Byron, and J. L. Sullivan. 1989. Severe herpesvirus infections in an adolescent without natural killer cells. N. Engl. J. Med. 320:1731-1735.[Medline]
8
8. Blazar, B., M. Patarroyo, E. Klein, and G. Klein. 1980. Increased sensitivity of human lymphoid lines to natural killer cells after induction of the Epstein-Barr viral cycle by superinfection or sodium butyrate. J. Exp. Med. 151:614-627.[Abstract/Free Full Text]
9
9. Bottino, C., R. Castriconi, D. Pende, P. Rivera, M. Nanni, B. Carnemolla, C. Cantoni, J. Grassi, S. Marcenaro, N. Reymond, M. Vitale, L. Moretta, M. Lopez, and A. Moretta. 2003. Identification of PVR (CD155) and nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J. Exp. Med. 198:557-567.[Abstract/Free Full Text]
10
10. Braud, V. M., D. S. Allan, C. A. O'Callaghan, K. Soderstrom, A. D'Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, L. L. Lanier, and A. J. McMichael. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391:795-799.[CrossRef][Medline]
11
11. Cerwenka, A., and L. L. Lanier. 2001. Natural killer cells, viruses and cancer. Nat. Rev. Immunol. 1:41-49.[CrossRef][Medline]
12
12. Cooper, M. A., T. A. Fehniger, S. C. Turner, K. S. Chen, B. A. Ghaheri, T. Ghayur, W. E. Carson, and M. A. Caligiuri. 2001. Human natural killer cells: a unique innate immunoregulatory role for the CD56^bright subset. Blood 97:3146-3151.[Abstract/Free Full Text]
13
13. Cosman, D., N. Fanger, L. Borges, M. Kubin, W. Chin, L. Peterson, and M. L. Hsu. 1997. A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 7:273-282.[CrossRef][Medline]
14
14. Decker, L. L., L. D. Klaman, and D. A. Thorley-Lawson. 1996. Detection of the latent form of Epstein-Barr virus DNA in the peripheral blood of healthy individuals. J. Virol. 70:3286-3289.[Abstract]
15
15. Diefenbach, A., and D. H. Raulet. 2001. Strategies for target cell recognition by natural killer cells. Immunol. Rev. 181:170-184.[CrossRef][Medline]
16
16. Fehniger, T. A., M. A. Cooper, G. J. Nuovo, M. Cella, F. Facchetti, M. Colonna, and M. A. Caligiuri. 2003. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood 101:3052-3057.[Abstract/Free Full Text]
17
17. Ferlazzo, G., and C. Munz. 2004. NK cell compartments and their activation by dendritic cells. J. Immunol. 172:1333-1339.[Free Full Text]
18
18. Ferlazzo, G., D. Thomas, S. L. Lin, K. Goodman, B. Morandi, W. A. Muller, A. Moretta, and C. Munz. 2004. The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic. J. Immunol. 172:1455-1462.[Abstract/Free Full Text]
19
19. Gudgeon, N. H., G. S. Taylor, H. M. Long, T. A. Haigh, and A. B. Rickinson. 2005. Regression of Epstein-Barr virus-induced B-cell transformation in vitro involves virus-specific CD8⁺ T cells as the principal effectors and a novel CD4⁺ T-cell reactivity. J. Virol. 79:5477-5488.[Abstract/Free Full Text]
20
20. Hochberg, D., T. Souza, M. Catalina, J. L. Sullivan, K. Luzuriaga, and D. A. Thorley-Lawson. 2004. Acute infection with Epstein-Barr virus targets and overwhelms the peripheral memory B-cell compartment with resting, latently infected cells. J. Virol. 78:5194-5204.[Abstract/Free Full Text]
21
21. Joncas, J., Y. Monczak, F. Ghibu, C. Alfieri, A. Bonin, G. Ahronheim, and G. Rivard. 1989. Brief report: killer cell defect and persistent immunological abnormalities in two patients with chronic active Epstein-Barr virus infection. J. Med. Virol. 28:110-117.[Medline]
22
22. Joseph, A. M., G. J. Babcock, and D. A. Thorley-Lawson. 2000. Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive B-cell subset of healthy tonsils. J. Virol. 74:9964-9971.[Abstract/Free Full Text]
23
23. Keating, S., S. Prince, M. Jones, and M. Rowe. 2002. The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules. J. Virol. 76:8179-8188.[Abstract/Free Full Text]
24
24. Kubin, M. Z., D. L. Parshley, W. Din, J. Y. Waugh, T. Davis-Smith, C. A. Smith, B. M. Macduff, R. J. Armitage, W. Chin, L. Cassiano, L. Borges, M. Petersen, G. Trinchieri, and R. G. Goodwin. 1999. Molecular cloning and biological characterization of NK cell activation-inducing ligand, a counterstructure for CD48. Eur. J. Immunol. 29:3466-3477.[CrossRef][Medline]
25
25. Laichalk, L. L., and D. A. Thorley-Lawson. 2005. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo. J. Virol. 79:1296-1307.[Abstract/Free Full Text]
26
26. Lee, N., D. R. Goodlett, A. Ishitani, H. Marquardt, and D. E. Geraghty. 1998. HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J. Immunol. 160:4951-4960.[Abstract/Free Full Text]
27
27. Lodoen, M. B., and L. L. Lanier. 2005. Viral modulation of NK cell immunity. Nat. Rev. Microbiol. 3:59-69.[CrossRef][Medline]
28
28. Lopez-Gonzalez, M. A., B. Sanchez, F. Mata, and F. Delgado. 1998. Tonsillar lymphocyte subsets in recurrent acute tonsillitis and tonsillar hypertrophy. Int. J. Pediatr. Otorhinolaryngol. 43:33-39.[CrossRef][Medline]
29
29. Lozzio, C. B., and B. B. Lozzio. 1975. Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45:321-334.[Abstract/Free Full Text]
30
30. Merino, F., C. Amesty, W. Henle, Z. Layrisse, N. Bianco, and P. Ramirez-Duque. 1986. Chediak-Higashi syndrome: immunological responses to Epstein-Barr virus studies in gene heterozygotes. J. Clin. Immunol. 6:242-248.[CrossRef][Medline]
31
31. Miyashita, E., B. Yang, G. Babcock, and D. Thorley-Lawson. 1997. Identification of the site of Epstein-Barr virus persistence in vivo as a resting B cell. J. Virol. 71:4882-4891.[Abstract]
32
32. Moretta, A., P. Comoli, D. Montagna, A. Gasparoni, E. Percivalle, I. Carena, M. G. Revello, G. Gerna, G. Mingrat, F. Locatelli, G. Rondini, and R. Maccario. 1997. High frequency of Epstein-Barr virus (EBV) lymphoblastoid cell line-reactive lymphocytes in cord blood: evaluation of cytolytic activity and IL-2 production. Clin. Exp. Immunol. 107:312-320.[CrossRef][Medline]
33
33. Morris, R. J., L. K. Chong, G. W. Wilkinson, and E. C. Wang. 2005. A high-efficiency system of natural killer cell cloning. J. Immunol. Methods 307:24-33.[CrossRef][Medline]
34
34. Morrison, T. E., A. Mauser, A. Wong, J. P. Ting, and S. C. Kenney. 2001. Inhibition of IFN-gamma signaling by an Epstein-Barr virus immediate-early protein. Immunity 15:787-799.[CrossRef][Medline]
35
35. Niedobitek, G., A. Agathanggelou, N. Steven, and L. S. Young. 2000. Epstein-Barr virus (EBV) in infectious mononucleosis: detection of the virus in tonsillar B lymphocytes but not in desquamated oropharyngeal epithelial cells. Mol. Pathol. 53:37-42.[Abstract/Free Full Text]
36
36. Orange, J. S., M. S. Fassett, L. A. Koopman, J. E. Boyson, and J. L. Strominger. 2002. Viral evasion of natural killer cells. Nat. Immunol. 3:1006-1012.[CrossRef][Medline]
37
37. Parolini, S., C. Bottino, M. Falco, R. Augugliaro, S. Giliani, R. Franceschini, H. D. Ochs, H. Wolf, J. Y. Bonnefoy, R. Biassoni, L. Moretta, L. D. Notarangelo, and A. Moretta. 2000. X-linked lymphoproliferative disease. 2B4 molecules displaying inhibitory rather than activating function are responsible for the inability of natural killer cells to kill Epstein-Barr virus-infected cells. J. Exp. Med. 192:337-346.[Abstract/Free Full Text]
38
38. Pudney, V. A., A. M. Leese, A. B. Rickinson, and A. D. Hislop. 2005. CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells. J. Exp. Med. 201:349-360.[Abstract/Free Full Text]
39
39. Qu, L., and D. T. Rowe. 1992. Epstein-Barr virus latent gene expression in uncultured peripheral blood lymphocytes. J. Virol. 66:3715-3724.[Abstract/Free Full Text]
40
40. Ressing, M. E., S. E. Keating, D. van Leeuwen, D. Koppers-Lalic, I. Y. Pappworth, E. J. H. J. Wiertz, and M. Rowe. 2005. Impaired transporter associated with antigen processing-dependent peptide transport during productive EBV infection. J. Immunol. 174:6829-6838.[Abstract/Free Full Text]
41
41. Ressing, M. E., D. van Leeuwen, F. A. Verreck, R. Gomez, B. Heemskerk, M. Toebes, M. M. Mullen, T. S. Jardetzky, R. Longnecker, M. W. Schilham, T. H. Ottenhoff, J. Neefjes, T. N. Schumacher, L. M. Hutt-Fletcher, and E. J. Wiertz. 2003. Interference with T cell receptor-HLA-DR interactions by Epstein-Barr virus gp42 results in reduced T helper cell recognition. Proc. Natl. Acad. Sci. USA 100:11583-11588.[Abstract/Free Full Text]
42
42. Ressing, M. E., D. van Leeuwen, F. A. Verreck, S. Keating, R. Gomez, K. Franken, T. H. M. Ottenhoff, M. Spriggs, T. N. Schumacher, L. M. Hutt-Fletcher, M. Rowe, and E. J. H. J. Wiertz. 2005. Epstein-Barr virus gp42 is posttranslationally modified to produce soluble gp42 that mediates HLA class II immune evasion. J. Virol. 79:841-852.[Abstract/Free Full Text]
43
43. Reyburn, H. T., O. Mandelboim, M. Vales-Gomez, D. M. Davis, L. Pazmany, and J. L. Strominger. 1997. The class I MHC homologue of human cytomegalovirus inhibits attack by natural killer cells. Nature 386:514-517.[CrossRef][Medline]
44
44. Robertson, M. J., K. J. Cochran, C. Cameron, J. M. Le, R. Tantravahi, and J. Ritz. 1996. Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukemia. Exp. Hematol. 24:406-415.[Medline]
45
45. Rooney, C. M., C. A. Smith, C. Y. C. Ng, S. Loftin, C. F. Li, R. A. Krance, M. K. Brennert, and H. E. Heslop. 1995. Use of gene-modified virus-specific T-lymphocytes to control Epstein-Barr virus-related lymphoproliferation. Lancet 345:9-13.[CrossRef][Medline]
46
46. Shilling, H. G., N. Young, L. A. Guethlein, N. W. Cheng, C. M. Gardiner, D. Tyan, and P. Parham. 2002. Genetic control of human NK cell repertoire. J. Immunol. 169:239-247.[Abstract/Free Full Text]
47
47. Sivori, S., D. Pende, C. Bottino, E. Marcenaro, A. Pessino, R. Biassoni, L. Moretta, and A. Moretta. 1999. NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human NK cells. Correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells. Eur. J. Immunol. 29:1656-1666.[CrossRef][Medline]
48
48. Speck, S. H. 2005. Regulation of EBV latency-associated gene expression, p. 403-427. In E. S. Robertson (ed.), Epstein-Barr virus. Caister Academic Press, Norfolk, United Kingdom.
49
49. Sutherland, C. L., N. J. Chalupny, K. Schooley, T. VandenBos, M. Kubin, and D. Cosman. 2002. UL16-binding proteins, novel MHC class I-related proteins, bind to NKG2D and activate multiple signaling pathways in primary NK cells. J. Immunol. 168:671-679.[Abstract/Free Full Text]
50
50. Tangye, S. G., J. H. Phillips, L. L. Lanier, and K. E. Nichols. 2000. Functional requirement for SAP in 2B4-mediated activation of human natural killer cells as revealed by the X-linked lymphoproliferative syndrome. J. Immunol. 165:2932-2936.[Abstract/Free Full Text]
51
51. Thorley-Lawson, D. 2005. EBV persistence and latent infection in vivo, p. 309-357. In E. S. Robertson (ed.), Epstein-Barr virus. Caister Academic Press, Norfolk, United Kingdom.
52
52. Tierney, R. J., N. Steven, L. S. Young, and A. B. Rickinson. 1994. Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. J. Virol. 68:7374-7385.[Abstract/Free Full Text]
53
53. Tomasec, P., V. M. Braud, C. Rickards, M. B. Powell, B. P. McSharry, S. Gadola, V. Cerundolo, L. K. Borysiewicz, A. J. McMichael, and G. W. Wilkinson. 2000. Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 287:1031-1033.[Abstract/Free Full Text]
54
54. Tomasec, P., E. C. Wang, A. J. Davison, B. Vojtesek, M. Armstrong, C. Griffin, B. P. McSharry, R. J. Morris, S. Llewellyn-Lacey, C. Rickards, A. Nomoto, C. Sinzger, and G. W. Wilkinson. 2005. Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat. Immunol. 6:181-188.[CrossRef][Medline]
55
55. Tortorella, D., B. E. Gewurz, M. H. Furman, D. J. Schust, and H. L. Ploegh. 2000. Viral subversion of the immune system. Annu. Rev. Immunol. 18:861-926.[CrossRef][Medline]
56
56. Vitale, C., L. Chiossone, G. Morreale, E. Lanino, F. Cottalasso, S. Moretti, G. Dini, L. Moretta, and M. C. Mingari. 2005. Human natural killer cells undergoing in vivo differentiation after allogeneic bone marrow transplantation: analysis of the surface expression and function of activating NK receptors. Mol. Immunol. 42:405-411.[CrossRef][Medline]
57
57. Vossen, M. T., E. M. Westerhout, C. Soderberg-Naucler, and E. J. Wiertz. 2002. Viral immune evasion: a masterpiece of evolution. Immunogenetics 54:527-542.[CrossRef][Medline]
58
58. Wang, E. C., B. McSharry, C. Retiere, P. Tomasec, S. Williams, L. K. Borysiewicz, V. M. Braud, and G. W. Wilkinson. 2002. UL40-mediated NK evasion during productive infection with human cytomegalovirus. Proc. Natl. Acad. Sci. USA 99:7570-7575.[Abstract/Free Full Text]
59
59. Welte, S. A., C. Sinzger, S. Z. Lutz, H. Singh-Jasuja, K. L. Sampaio, U. Eknigk, H. G. Rammensee, and A. Steinle. 2003. Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein. Eur. J. Immunol. 33:194-203.[CrossRef][Medline]
60
60. Williams, H., K. McAulay, K. F. Macsween, N. J. Gallacher, C. D. Higgins, N. Harrison, A. J. Swerdlow, and D. H. Crawford. 2005. The immune response to primary EBV infection: a role for natural killer cells. Br. J. Haematol. 129:266-274.[CrossRef][Medline]
61
61. Wills, M. R., O. Ashiru, M. B. Reeves, G. Okecha, J. Trowsdale, P. Tomasec, G. W. Wilkinson, J. Sinclair, and J. G. Sissons. 2005. Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis. J. Immunol. 175:7457-7465.[Abstract/Free Full Text]
62
62. Wilson, A. D., and A. J. Morgan. 2002. Primary immune responses by cord blood CD4⁺ T cells and NK cells inhibit Epstein-Barr virus B-cell transformation in vitro. J. Virol. 76:5071-5081.[Abstract/Free Full Text]
63
63. Wooden, S. L., S. R. Kalb, R. J. Cotter, and M. J. Soloski. 2005. Cutting edge: HLA-E binds a peptide derived from the ATP-binding cassette transporter multidrug resistance-associated protein 7 and inhibits NK cell-mediated lysis. J. Immunol. 175:1383-1387.[Abstract/Free Full Text]
64
64. Yewdell, J. W., and A. B. Hill. 2002. Viral interference with antigen presentation. Nat. Immunol. 3:1019-1025.[CrossRef][Medline]
65
65. Young, L. S., R. Lau, M. Rowe, G. Niedobitek, G. Packham, F. Shanaham, D. T. Rowe, D. Greenspan, J. S. Greenspan, A. B. Rickinson, and P. J. Farrell. 1991. Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral "hairy" leukoplakia. J. Virol. 65:2868-2874.[Abstract/Free Full Text]
66
66. Zeidler, R., G. Eissner, P. Meissner, S. Uebel, R. Tampe, S. Lazis, and W. Hammerschmidt. 1997. Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10. Blood 90:2390-2397.[Abstract/Free Full Text]

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