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The Journal of Immunology, 2004, 173: 321-331.
Copyright © 2004 by The American Association of Immunologists

Epstein-Barr Virus Promotes Human Monocyte Survival and Maturation through a Paracrine Induction of IFN-{alpha}

Shahram Salek-Ardakani1, Stephen A. Lyons and John R. Arrand

Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Withington, Manchester, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of monocytes and macrophages during EBV infection is not clear. The interaction of EBV with human monocytes was investigated in terms of cell survival and morphological and phenotypic changes to gain a better understanding of the role of these cells during EBV infection. We show that EBV infection of PBMCs rescues monocytes from undergoing spontaneous apoptosis and dramatically enhances their survival. Results obtained with heat-inactivated virus, neutralizing anti-EBV mAb 72A1 and recombinant gp350, suggest that enhancement of viability by EBV requires both infectious virus and interaction between gp350 and its receptor. IFN-{alpha} either secreted within 24 h from PBMCs upon infection with EBV or exogenously added to unstimulated monocytes inhibited spontaneous apoptosis, indicating that induction of IFN-{alpha} is an early important survival signal responsible for the delay in the apoptosis of monocytes. EBV infection also induced acute maturation of monocytes to macrophages with morphological and phenotypic characteristics of potent APCs. Monocytes exposed to EBV became larger in size with increased granularity and expressed considerably higher levels of membrane HLA classes I and II, ICAM-1, CD80, CD86, and CD40 compared with uninfected cultures. These observations provide the first immunoregulatory links among EBV, IFN-{alpha}, and monocyte survival and maturation and importantly raise the possibility that these cells may serve as a vehicle for the dissemination of the virus as well as being active participants in eliciting anti-EBV T cell responses during acute infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epstein-Barr virus is a ubiquitous human {gamma}-herpes virus, present in ~90% of the world population. Primary EBV infection at an early age is usually asymptomatic, and it is only in more developed countries where such infection is delayed that disease manifests as infectious mononucleosis (1, 2). In either case, the infected individuals continue to harbor the virus without further expression of clinical symptoms. Under certain conditions EBV infection may also be associated with the development of Burkitt’s lymphoma, Hodgkin’s disease, undifferentiated nasopharyngeal carcinoma, immunoblastic lymphomas arising in immunocompromised individuals, and T and NK cell lymphomas (1, 2).

Monocytes are highly versatile cells playing crucial roles in the maintenance of immune homeostasis. These cells are released into the bloodstream from the bone marrow and, in the absence of specific survival signals, are programmed to undergo apoptosis in 24–48 h (3, 4). Shortly after viral infections, circulating monocytes transmigrate through vascular endothelium and localize in infected tissues, where they undergo differentiative events controlled by the specific microenvironment they enter. Here, extracellular stimuli, including cytokines released from infected orinflammatory cells, adhesion/costimulatory molecules, and viral products determine the differentiation of monocytes into functionally distinct, long-lived tissue macrophages (4, 5). In addition, CD14+ monocytes can be induced to differentiate to dendritic cells (DCs)2 both in vitro and in vivo, indicating divergent pathways for monocyte differentiation (6, 7, 8). These cells provide an innate, Ag-nonspecific, first line of defense against infection mostly by phagocytosis and generation of degradative enzymes and reactive oxygen metabolites (9). Furthermore, they play a pivotal role in the induction and regulation of specific antiviral T cell responses through binding of antigenic-MHC complexes to specific TCR molecules and release of immunomodulatory cytokines (10, 11, 12).

Evidence for an interaction between EBV and monocytes is growing. The EBV receptor, CR2/CD21, is expressed on a minor (~20%) subset of human monocytes (13, 14), and direct binding of EBV to monocytic cells has been demonstrated (15). Moreover, EBV DNA and proteins have been detected in monocytic cell lines obtained from bone marrow of children suffering from maturation defects of hemopoiesis (16) and in cultured macrophages obtained from patients with benign or malignant neoplasms (17). Consistent with these observations, Savard et al. (18) demonstrated the ability of EBV to infect and replicate in a subpopulation of purified human peripheral blood monocytes, as revealed by the detection of EBNA-1 and immediate-early, early, and late lytic mRNA transcripts. Taken together, these studies suggest that EBV may interact with monocytes in vivo and modulate their physiological activities. However, in the absence of a good animal model for EBV infection, the precise role of monocytes and macrophages in the EBV life cycle has been difficult to ascertain.

Several lines of evidence indicate that the interaction between EBV and monocytes and macrophages may have important consequences for both the host and the virus. Interaction of EBV with monocytes has been shown to inhibit the synthesis of the pleiotropic cytokine TNF-{alpha} (15) and PGE2, both of which exhibit potent antiviral activity (19). Conversely, EBV induces the synthesis of GM-CSF (20) and the formation of important lipid mediators of inflammation, such as leukotrienes B4 and C4, by a mechanism involving the major viral envelope gp350 (21). EBV infection of purified monocytes and macrophages, respectively, results in the suppression or enhanced phagocytic activity by these same cells, indicating that the outcome of infection is dependent upon the monocyte maturation state (17, 18). Recently, Li et al. (22) demonstrated that EBV infection inhibits the development of DCs by promoting apoptosis of their monocyte precursors cultured in the presence of GM-CSF plus IL-4. Thus, modulation of monocyte survival and maturation may represent an important strategy used by EBV to interfere with the virus-specific immune responses.

To learn about the potential role of monocytes during EBV infection, the present study was initiated to characterize the basic interaction of EBV with these cells in terms of cell survival and morphological and phenotypic changes. Given that monocyte survival and maturation are critically regulated by cytokines (4, 23, 24, 25), we wanted to use an in vitro human PBMC culture system, rather than purified cells or cell lines, because it has been shown to accurately recapitulate the cytokine response to EBV seen during an in vivo infection (26, 27, 28, 29, 30, 31, 32, 33). Although EBV infection had little or no effect on maturation and survival of purified monocytes, it dramatically enhanced monocyte survival in the PBMC system and rapidly induced their maturation to macrophages with a morphology and phenotype characteristic of potent APCs. Our results indicate that prompt (within 24 h) induction of IFN-{alpha} from PBMCs upon infection with EBV is critical for its monocyte survival-enhancing effect. These findings provide the first immunoregulatory links among EBV, IFN-{alpha}, and monocyte survival and maturation. Importantly, they raise the possibility that these cells may serve as a vehicle for the dissemination of the virus as well as being active participants in eliciting anti-EBV T cell responses during acute infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cytokines and reagents

Recombinant human TNF-{alpha}, IFN-{alpha}, IFN-{gamma}, IL-1{beta}, and GM-CSF were all obtained from PeproTech (London, U.K.). LPS and PMA were obtained from Sigma-Aldrich (St. Louis, MO). The recombinant gp350 (rgp350) preparation used was a gift from Dr. G. Roberts (British Biotechnology, Oxford, U.K.). When examined on silver-stained 5% SDS-polyacrylamide gel, the protein was of the expected size (data not shown), and its immunological activity was similar to that of authentic EBV-gp350 (34).

Preparation of concentrated stocks of EBV virions

Virions were prepared from culture supernatants of the B95-8 cell line (35). Virus replication was induced by tightening the cell culture flask tops and incubating for 7 days at 37°C in 5% CO2. The culture supernatant was centrifuged (400 x g, 15 min), filtered (using 0.45-µm bottletop filters; Falcon; BD Biosciences, Franklin Lakes, NJ) to remove cell debris and concentrated 100-fold using a Hollow Fiber & TurboTube ultrafiltration cartridge with a molecular mass exclusion of 500 kDa (A/G Technology). Concentrated viral preparations were resuspended in RPMI 1640, aliquoted, and stored at –80°C. Viral titers were evaluated and adjusted to 2 x 107 transforming units (TFU)/ml, as previously described (36). In some experiments EBV virions were inactivated by heating at 56°C for 1 h. Heat inactivation completely abolished the ability of the virus to transform cord blood mononuclear cells. The neutralizing mAb 72A1 (provided by Dr. A. Morgan, University of Bristol, Bristol, U.K.) raised against the major EBV viral envelope gp350 was used to evaluate the specificity of the response obtained with EBV.

Isolation of mononuclear cells from peripheral blood

Peripheral blood from healthy donors was collected into tubes containing preservative-free heparin (CP Pharmaceuticals, Wrexham, U.K.) and processed within 1 h of withdrawal. Mononuclear cells were isolated as described previously (37). Where indicated, monocytes were isolated from PBMCs using a FACSVantage (BD Biosciences, Mountain View, CA) cell sorter based on the forward (FSC) and side (SSC) scatter characteristics of these cells. The purity of the monocytes was confirmed by staining the enriched cells with R-PE-Cy5-conjugated mouse anti-human mAb against the monocyte/macrophage-specific marker, CD14, and was >98% (Fig. 5).



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  		FIGURE 5. EBV enhances monocyte survival through an IFN-{alpha}-dependent mechanism. A–D, Purification of monocytes by cell sorting. A, Typical dot plot of human PBMCs. The intensity of forward scatter (FSC; x-axis) is plotted against the intensity of right angle scatter (SSC; y-axis) for each cell. Each dot represents one cell. B and C, Monocytes (gated region R1) were isolated from PBMCs by two rounds of cell sorting based on their FSC and SSC characteristics. Numbers given in the upper right corners of the dot plots indicate the percentage of monocytes after each round of cell sorting. D, The purity of monocytes was confirmed by staining with PE-Cy5-conjugated anti-mAbs against the monocyte/macrophage-specific marker, CD14. E, Purified monocytes were cultured for 3–10 days in growth medium alone or with infectious EBV (0.2 x 105 TFU/ml). After each incubation period, the indicated cells were harvested and double stained with PE-Cy5-conjugated anti-CD14 and FDA. The absolute numbers of CD14+/FDA+ cells were determined by FACS and are presented as the mean of triplicate wells ± SD from two independent experiments. F and G, Supernatants from 1- or 3-day EBV or mock-infected cultures were pooled, filtered, and transferred to uninfected monocytes. FDA and CD14 double-positive cells were counted on day 3. F, The absolute numbers of CD14+/FDA+ cells were determined by FACS and are presented as the mean of triplicate wells ± SD from four independent experiments. Background numbers of monocytes cultured in medium alone (Expt. 1, 18,201; Expt. 2, 19,094; Expt. 3, 6,859; Expt. 4, 3,980) were subtracted from the data shown. Infectious EBV (0.2 x 105 TFU/ml) was used as a positive control. H, PBMCs were cultured in medium alone or with increasing concentrations of IFN-{alpha}, IFN-{gamma}, IL-1{beta}, and GM-CSF as indicated. After 3 days, the absolute numbers of CD14+/FDA+ cells were determined by FACS and are presented as the mean of triplicate wells ± SD from two independent experiments. I, Infectious EBV (0.2 x 105 TFU/ml) was used as a positive control. J, PBMCs were cultured in medium alone; EBV (0.2 x 105 TFU/ml); EBV plus saturating concentrations of anti-IFN-{alpha}, anti-IFN-{gamma}, anti-IL-1{beta}, and anti-GM-CSF neutralizing Abs; EBV plus anti-IFN-{alpha} neutralizing Abs added to the culture 1 day postinfection; EBV plus rIFN-{alpha} (0.2 ng/ml); or IFN-{alpha} (0.2 ng/ml) alone. After 3 days, the absolute numbers of CD14+/FDA+ cells were determined by FACS and are presented as the mean of triplicate wells ± SD from two independent experiments. K, Purified monocytes were cultured in medium alone or in the presence of EBV, EBV plus rIFN-{alpha} (0.2 ng/ml), or IFN-{alpha} (0.2 ng/ml) alone. After 3 days, the absolute numbers of CD14+/FDA+ cells were determined by FACS and are presented as the mean of triplicate wells ± SD from two independent experiments.

 
Determination of cell survival

PBMCs (1 x 106 cells/ml) or purified monocytes (5 x 105 cells/ml) were suspended in HEPES (25 mM)-buffered RPMI 1640 containing 100 U/ml penicillin and 100 µg/ml streptomycin, 2 mM L-glutamine, and 10% FCS (all from Life Technologies, Gaithersburg, MD). PBMC suspension (0.1 ml) was transferred to each well of a 96-well, U-bottom tissue culture plate (Nunc, Naperville, IL). Cells were infected with 10-fold serial dilutions of concentrated EBV stock in a final volume of 200 µl. PBMCs incubated with TNF-{alpha}, LPS, and PMA were used as positive controls for induction of monocyte/macrophage apoptosis. The plates were incubated at 37°C in 5% CO2 for 1–30 days with weekly feeding by removing 100 µl of spent medium and replacing with fresh medium. After the required incubation period, cells were pelleted by centrifugation at 200 x g for 3 min and resuspended in 200 µl of PBS. The viability of monocyte/macrophages was determined by direct immunofluorescence and was analyzed by flow cytometry (see below).

Flow cytometric assessment of cell survival

The number of viable or apoptotic monocytes/macrophages was determined on the basis of cell surface staining by the monocyte/macrophage-specific marker CD14 and the differential uptake of fluorescein diacetate (FDA; Sigma-Aldrich) using a modification of the flow cytometric method described by Jones et al. (38) and Ross et al. (39). Briefly, after incubation, cells were washed three times with PBS, stained with CD14 as previously described (37), and resuspended in 200 µl of PBS. For staining, FDA stock solution (5 mg/ml in acetone) was added to each well at a final concentration of 100 ng/ml in PBS. Cells were incubated for 10 min at room temperature and transferred to 5-ml tubes, which were kept on ice until further analysis by FACS. Microscopic examination of each well confirmed that all cells were removed from the plates. Monocytes and macrophages were identified based on CD14 staining using a FACScan flow cytometer. Viable cells were identified by their ability to take up FDA and convert it to fluorescein, which accumulates inside viable cells with intact plasma membrane integrity and exhibits green fluorescence when excited with blue light. Dead cells are negative for green fluorescence. The validity of this method of viability measurement was confirmed by comparison with other assays of apoptosis, including the FDA/propidium iodide method (39), propidium uptake (cells were incubated in 0.05 mg/ml propidium iodide in PBS for 15 min on ice before analysis), and annexin V staining (BD Pharmingen, San Diego, CA; cells stained for cytometry were incubated for 15 min in 10 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2, and the manufacturer’s recommended dilution of annexin V-PE and 7-aminoactinomycin D (7-AAD)). Each measure of apoptosis gave similar values (Fig. 2B and data not shown). The number of viable cells (CD14+/FDA+) per milliliter of culture was determined by making a timed count of cells in different quadrants, then calculating viable cells per milliliter by dividing the number of cells in each quadrant per unit time by the sample flow rate as previously described by Ross et al. (39). Final data are expressed as the number of viable cells per milliliter of culture medium.



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  		FIGURE 2. EBV enhances monocyte survival by an antiapoptotic mechanism. A, PBMCs were infected with EBV (0.2 x 105 TFU/ml) or mock-infected and harvested at the time points indicated. Apoptotic monocytes were identified by staining with FITC-conjugated anti-CD14 and PE-conjugated annexin V and 7-AAD. The percentages of apoptotic and viable monocytes within PBMCs were determined by FACS and are presented in the upper and lower left regions of each dot plot, respectively. 7-AAD-positive cells (dead cells) were excluded from the analysis. Quadrant settings, distinguishing positive from background fluorescence, were determined by staining with isotype-matched control mAbs. B, Comparison of FDA and annexin V staining for determination of viable monocyte counts. PBMCs were infected with EBV for 3 days, then stained with either the combination of anti-CD14 and FDA or anti-CD14, annexin V, and 7-AAD as indicated. The total number of viable (FDA+, annexin V–, 7-AAD–) monocytes (CD14+) was then determined by differential staining of FDA, annexin V, and 7-AAD, and the results were analyzed by FACS. The results show that both methods for counting viable (FDA+, annexin V–, 7-AAD–) monocyte numbers gave comparable (within 10%) counts.

 
Neutralizing experiments

PBMCs (1 x 106 cells/ml) were cultured for 3 days in growth medium alone (medium), EBV (0.2 x 105 TFU/ml), or EBV plus saturating concentrations of neutralizing Abs against IFN-{alpha} (15 µg/ml), IFN-{gamma} (15 µg/ml), GM-CSF (15 µg/ml), and IL-1{beta} (15 µg/ml; all obtained from PeproTech). After each incubation period (see figure legends for details), cells were harvested and double stained with PE-Cy5-conjugated anti-CD14 and FDA. The percentages of CD14+/FDA+ cells within PBMCs were determined by FACS and are presented as the mean ± SD of triplicate wells.

Phenotypic and morphological analysis of monocytes/macrophages

PBMCs (2 x 106 cells/ml) were infected with EBV (0.2 x 105 TFU/ml) in a final volume of 200 µl in a 96-well plate (Falcon). After 1 day in culture, cells were harvested, stained for the particular cell surface marker under investigation, and analyzed by FACS as described previously (37). Abs used for staining cell surface markers were FITC-conjugated mouse anti-human HLA-A, -B, and -C (HLA class I; G46-2.6), CD40, CD48 (TU145), and CD86 (2331(FUN-1)) and R-PE-conjugated mouse anti-human CD80 (BB1; BD Pharmingen); FITC-conjugated mouse anti-human HLA-DP, -DQ, -DR (HLA-class II; CR3/43) and CD54 (ICAM-1; 6.5B5) and R-PE-conjugated mouse anti-human CD19 (HD37; DAKO, Carpinteria, CA); FITC-conjugated mouse anti-human CD102 (ICAM-2; B-T1) and R-PE-Cy5-conjugated mouse anti-human CD14 (TUK4; Serotec, Oxford, U.K.); and FITC-conjugated mouse anti-human CD50 (ICAM-3; Cal 3.10; R&D Systems, Minneapolis, MN).

Control of endotoxin levels

All cell culture reagents used were either certified as low in endotoxin when purchased or were ensured to be low in endotoxin by the Pyrogenet-5000 turbidimetric Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) using the Kinetic-QCL reader and WinKQCL software (BioWhittaker).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EBV infection of PBMCs enhances monocyte survival by an antiapoptotic mechanism

We used cultured PBMCs to investigate the effect of EBV infection on monocyte survival. The viability of monocytes within PBMCs was quantified by measuring FDA uptake in CD14+ (monocyte/macrophage-specific cell surface marker) cells using FACS analysis. Firstly, control experiments were conducted to ensure that the culture conditions and viability assay were appropriate. In accordance with previous findings, addition of high concentrations of TNF-{alpha} (40), LPS (41), or PMA (42) at the beginning of the culture period reduced the numbers of viable monocytes (CD14+/FDA+) by ~32, ~57, and ~98%, respectively (Fig. 1, A and B). It is now well established that in the absence of exogenous stimuli, cultured peripheral blood monocytes undergo apoptosis within 24–72 h (3, 4, 23). Consistent with these observations, the percentage and the absolute numbers of freshly isolated monocytes cultured in the absence of a survival stimulus (medium alone) rapidly decreased from 15 ± 0.08% (52,604 ± 159 cells/ml) on day 1 to 3 ± 0.3% (17,715 ± 2,477 cells/ml) of total PBMCs on day 3 (Fig. 1, C and D). EBV infection of PBMCs rescued monocytes from undergoing spontaneous apoptosis between days 1 and 3 in culture compared with uninfected cultures (Fig. 1, C and D). EBV titers as low as 0.02 x 105 TFU/ml enhanced the numbers of monocytes/macrophages from 17,715 ± 2,477 to 41,353 ± 1,322 cells/ml, an increase of 2.33-fold. Maximal viability, corresponding to an increase of 2.6- to 2.9-fold, was attained with virus titers between 0.2 and 2 x 105 TFU/ml. The results depicted in Fig. 1E and Table I show that EBV enhanced monocyte survival at a range of cell densities and in PBMCs obtained from multiple donors, respectively. However, the level of protection varied between individual donors, with increases from 2- to 7-fold increases compared with control cultures.



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  		FIGURE 1. EBV infection promotes human monocyte survival. A, PBMCs were cultured in medium alone or in the presence of TNF-{alpha} (20 ng/ml), LPS (1 µg/ml), or PMA (0.1 ng/ml). After 1 day, cells were harvested, and the numbers of viable monocytes/macrophages were determined by staining with PE-Cy5-conjugated anti-CD14 mAb and FDA, followed by FACS analysis. The numbers in the upper left quadrant of each scatter plot indicate the percentage of the total PBMC population identified as viable monocytes/macrophages. B, The average viable monocyte/macrophage counts from three independent experiments presented as a histogram. C and D, PBMCs were cultured for 1 or 3 days in growth medium alone or in the presence of increasing titers of EBV as indicated, after which cells were harvested and the percentage (C) and absolute numbers (D) of viable monocytes were determined by FACS. E, PBMCs were cultured at three different cell densities (0.5–2 x 106 cells/ml) in the presence or the absence of EBV (0.2 x 105 TFU/ml). After 3 days, cells were harvested and stained with anti-CD14 mAbs and FDA, and the absolute number of viable monocytes was determined by FACS.

 

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  		Table I. EBV infection of PBMCs enhances monocyte survivala

 
To confirm that monocytes were undergoing an apoptotic mode of death, we performed studies with annexin V as a marker for apoptotic cells. PBMCs were cultured in the presence or the absence of EBV for 1–3 days as indicated, after which cells were harvested and double stained with anti-CD14 mAb and annexin V, and results were analyzed by FACS. In uninfected cultures the percentage of CD14+/annexin V+ cells (apoptotic cells) increased in a time-dependent manner from 36% on day 1 to 75% on day 3 (Fig. 2). These results are consistent with our data (Fig. 1, C and D) showing a decrease in the number of viable monocytes (CD14+/FDA+) between days 1 and 3 in culture. EBV infection was shown to reduce the percentage of monocytes (CD14+/annexin V+) undergoing spontaneous apoptosis at all time points examined, which is again in agreement with the data obtained with FDA uptake assessment of cell viability (Fig. 1, C and D). As a more direct comparison of FDA and annexin V/7-AAD staining for assessment of monocyte viability, PBMCs were infected with EBV for 3 days, and then cells were stained with anti-CD14 plus FDA or anti-CD14 plus annexin V and 7-AAD. Total numbers of viable (FDA+, annexin V/7-AAD–) monocytes from several samples were then determined based on differential staining of FDA, annexin V, and 7-AAD, and the results were analyzed by FACS. Fig. 2B shows that the number of viable monocytes obtained by FDA staining is comparable (within 10%) to those determined with annexin and 7-AAD staining. Taken together, these results indicate that EBV infection of PBMCs markedly enhances monocyte survival by reducing the spontaneous apoptosis induction normally seen in unstimulated monocytes in culture.

EBV infection of PBMCs markedly extends the longevity of monocytes

The above results suggested that EBV infection of PBMCs for 3 days was sufficient to confer longer life on monocytes. We conducted further CD14 and FDA labeling experiments to determine how long this protection lasted (Fig. 3). Viable monocytes constituted 10% (37,971 ± 1,506 cells/ml) of the total PBMC population on day 0. In untreated PBMCs the percentage of CD14+/FDA+ cells declined progressively with increasing time in culture, reaching <3% of total PBMCs on day 3 (Fig. 3). In contrast, infection of PBMCs with infectious EBV markedly increased the numbers of monocytes at each time point examined compared with control cells cultured in medium alone or heat-inactivated EBV. In EBV-infected cultures 50% viability was still detectable even 9 days postinfection. These results indicate that EBV infection of PBMCs supports long term survival of monocytes. All additional experiments were examined at 3 days postinfection unless indicated otherwise.



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  		FIGURE 3. EBV infection supports long term survival of monocytes. A, PBMCs were cultured for 1–30 days in growth medium alone, medium plus EBV (0.2 x 105 TFU/ml), or medium plus heat-inactivated EBV (H/IN). Viable monocytes were identified as being positive for both CD14 and FDA staining. The percentages of viable monocytes/macrophages within PBMCs were determined by FACS and are presented in the upper left region of each dot plot. B, Absolute numbers of viable monocytes/macrophages within PBMCs after each culture period indicated. The profiles shown are the mean ± SD of triplicate wells, and the experiments were repeated three times.

 
Enhancement of monocyte survival is not due to soluble factors/contaminants

In the light of the above observations it became important to show that the enhancement of monocyte survival seen in our assays was due to EBV particles rather than some hypothetical contaminant in the virus preparation. The EBV used in this study was concentrated from the culture medium of B95-8 cells. Although we avoided the use of phorbol esters in virus induction, it was possible that other potentially stimulatory materials had been concentrated together with the virus. This possibility was examined in two ways. Firstly, we conducted an experiment in which PBMCs were cultured for 3 days in medium alone or in the presence of virus prepared in our laboratory or virus prepared at University of Birmingham (Birmingham, U.K.). Both EBV preparations were able to enhance the survival of monocytes equally well (Fig. 4A). Secondly, a mock virus inoculum was prepared by passing the virus stock three times through a sterile, nonpyrogenic, 0.2-µm pore size filter. In each of four separate experiments in which EBV enhanced the survival of monocytes on day 3, the effect was completely eliminated by filtration of the virus stock (Fig. 4A). These results exclude the possibility that the effect of EBV on monocyte survival is due to soluble factors present in our virus preparation.



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  		FIGURE 4. Infectious EBV is required for the enhancement of monocyte survival and occurs via a mechanism involving gp350. A, PBMCs were infected with the following EBV stocks: virus prepared in our laboratory, (EBV (SS); 0.2 x 105 TFU/ml), virus prepared at University of Birmingham (EBV[AR]), or an EBV stock passed three times through a 0.2-µm pore size filter (0.2 µm). B, PBMCs were infected with EBV alone or in combination with a neutralizing mAb recognizing the major EBV gp350 (72A1). C, PBMCs were infected with EBV or exposed to increasing concentrations of purified rgp350 (0.1, 1, and 10 µg/ml) as indicated. For each experiment, viable monocyte counts were obtained on day 3. Results from medium only controls are also shown. For histograms, the background numbers of monocytes (cultured in medium alone) were subtracted from the monocyte counts in treated samples. The profiles shown are the mean ± SD of triplicate wells, and the experiments were repeated three times.

 
EBV-induced enhancement of monocyte survival occurs via a mechanism involving the major viral envelope gp350

To determine whether the effects seen on the monocyte population were due to a specific interaction of EBV with its cell surface receptor, experiments were conducted using a virus-neutralizing mAb 72A1 under the assay conditions outlined above. mAb 72A1 specifically binds to the major viral envelope gp350 and has been previously shown to block the attachment of EBV to the EBV receptor CR2/CD21 on B cells (43). Prior treatment of EBV with anti-gp350 mAb reduced the virus-induced increase in monocyte viability by ~75% (Fig. 4B), implying that the survival enhancing effect of EBV is mediated mostly through gp350. This raised the possibility that binding of gp350 to CD21/CR2 could alone promote monocyte survival. To test this possibility, PBMCs were cultured in the presence or the absence of infectious EBV or increasing concentrations of rgp350, and the viability of monocytes was determined as previously described. Little or no increase in viability was detected in cells exposed to rgp350 at concentrations of 0.1 and 1 µg/ml (Fig. 4C). However, at the highest concentration (10 µg/ml), a modest increase in the percentage (from 2.1 to 4.1%) and absolute numbers (from 7,908 ± 138 to 11,691 ± 94 cells/ml) of viable monocytes was detected compared with cells incubated with infectious EBV (Fig. 4C). Therefore, although the promotion of monocyte survival requires the interaction of EBV with its receptor, it is unlikely that the gp350 vs CD21/CR2 interaction alone can account for the full extent of this phenomenon.

EBV increases monocyte survival through an IFN-{alpha}-dependent mechanism

In principle, EBV-induced enhancement of monocyte survival could either be the result of direct action of the virus on the cells or be mediated by cytokines acting in an autocrine or paracrine fashion. We conducted experiments using purified monocytes to determine whether EBV can enhance monocyte survival by directly interacting with these cells. Monocytes were enriched to >98% from PBMCs by two rounds of cell sorting after gating on monocytes based on their FSC and SSC characteristics (Fig. 5, A–D). Purified monocytes were then cultured in the presence or the absence of EBV for 3–10 days as indicated, after which cellular viability was determined by CD14 and FDA staining. The survival-enhancing effect of EBV on purified monocytes was significantly reduced compared with its effect on monocytes present within PBMC cultures (compare Fig. 5E with Fig. 3B). These results imply that soluble factors produced by other cell types present within PBMCs are largely responsible for the EBV-induced enhancement of monocyte survival. To test this possibility, two identical sets of PBMCs were infected or mock infected with EBV for 1 or 3 days. After each incubation period, cell culture supernatants from each EBV-infected or mock-infected well were individually collected, pooled, and filtered to remove virus, cells, and debris particles. Supernatants were then transferred to uninfected PBMCs, and monocyte viability was determined after 3 days by FACS. Infectious EBV was used as a positive control in all experiments. The data presented in Fig. 5, F and G, clearly show that supernatants collected from EBV-infected cultures either 1 or 3 days postinfection can transfer (on the average, by 50 and 70%, respectively) the survival-enhancing effect of the infectious virus on monocytes. These results suggest that EBV infection of PBMCs rapidly (within 24 h) induces the release of one or more soluble factors that are responsible for preventing the spontaneous apoptosis seen in unstimulated monocytes.

Apoptosis of monocytes can be prevented by a number of proinflammatory cytokines, including IFN-{alpha} (44), IFN-{gamma} (4, 24), IL-1{beta} (24, 45), and GM-CSF (46), all of which are secreted as a result of EBV infection of PBMCs (27, 29, 30, 31). First we confirmed the ability of these cytokines to promote monocyte survival in our PBMC system (Fig. 5H). Infectious EBV was used as a positive control (Fig. 5I). PBMCs were cultured for 3 days in the presence or the absence of increasing concentrations of recombinant cytokines, after which monocyte viability was determined by FACS. As shown in Fig. 5H, and in accordance with previously published data, all cytokines examined were capable of preventing the spontaneous apoptosis of monocytes in a concentration-dependent manner, with the most potent being IFN-{alpha}>GM-CSF>IFN-{gamma}>IL-1{beta}. Significantly, very low concentrations of IFN-{alpha} (0.02–0.2 ng/ml) and GM-CSF, but not IFN-{gamma} or IL-1{beta}, were shown to enhance the viability of monocytes to similar levels as those seen with infectious virus (compare Fig. 5, H and I).

To determine the identity of the soluble factor(s) responsible for the apoptosis-preventing effect of EBV, additional experiments were performed in which PBMCs were infected with EBV in the presence or the absence of saturating concentrations of neutralizing Abs for different cytokines on day 0, and cell viability was determined 3 days later by FACS. Fig. 5J shows that addition of anti-IFN-{alpha}-neutralizing Ab at the beginning of the culture period reduced the antiapoptotic effect of EBV by an average of 76% from days 1 to 3, whereas anti-IFN-{gamma}-, anti-IL-1{beta}-, or anti-GM-CSF-neutralizing Abs had little or no effect, indicating that virally induced IFN-{alpha} is specifically involved in this phenomenon. Next we assessed whether IFN-{alpha} has a direct effect on monocyte survival. Purified monocytes were cultured in the presence or the absence of EBV, IFN-{alpha}, and the combinations indicated in Fig. 5. After 2 days, cell viability was determined by FACS. Consistent with the data in Fig. 5E, EBV alone had little or no effect on the survival of purified monocytes, whereas IFN-{alpha} almost completely inhibited the spontaneous apoptosis seen in unstimulated monocytes (Fig. 5K). Addition of EBV to IFN-{alpha}-treated monocytes had no effect on the survival of monocytes compared with cells cultured in the presence of IFN-{alpha} alone. Taken together, these results imply that the enhancement of monocyte survival by IFN-{alpha} is a highly specific mechanism that is rapidly induced after EBV infection of PBMCs.

EBV infection of PBMCs induces profound morphological and phenotypic changes in monocytes

As a number of viruses, including HIV, measles virus, CMV, and HSV, have been shown to differentially regulate monocyte activation and maturation leading to either immune suppression orhyperactivation (47, 48, 49), we also investigated the effects of EBV on the morphology and phenotype of monocytes. PBMCs were cultured in the presence of EBV for 1–17 days or left unstimulated. On day 1, the morphological dot plot reproducibly revealed two distinct cell populations differing in size (FSC) and granularity (SSC; Fig. 6A). Cells in the gated region (R) were considered to represent monocytes/macrophages based on their FSC and SSC characteristics and expression of CD14 (Fig. 6B). Lymphocytes were smaller in size and were negative for CD14. During the first 3 days of culture, EBV infection of PBMCs resulted in a dramatic increase in size (FSC) of CD14+ cells, which continued in a time-dependent manner, reaching a maximum level on day 7 (Fig. 6C). Moreover, EBV infection of PBMCs increased the granularity of macrophages by 4-fold between days 1 and 17, SSC of 180 (± 5.68) to 721 (±1.48) respectively (Fig. 6D).



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  		FIGURE 6. Kinetics of EBV-induced morphological changes in monocytes. PBMCs were cultured in the presence or the absence of EBV (0.2 x 105 TFU/ml) for 1–17 days as indicated. After each culture period shown, cells were harvested, and their morphology was analyzed by FACS and presented as dot plots and histograms. A, The intensity of FSC (x-axis) is plotted against the intensity of right angle scatter (SSC; y-axis) for each cell. Each dot represents one cell. The profiles shown are representative of two independent experiments. B, After each culture period indicated, cells were harvested and stained with PE-Cy5-conjugated anti-CD14, and the percentage of monocytes (CD14+ cells) within each gated area was determined by FACS as described in Materials and Methods. The size (FSC; C) and granularity (right angle scatter (SSC); D) for each CD14+ cell are presented as histograms.

 
Next, we examined the effect the effect of EBV on the phenotype of monocytes. PBMCs were exposed for 1 day to EBV, and the expression of several cell surface Ags known to be markers of monocyte maturation and activation was determined by direct immunofluorescence and analyzed by FACS. EBV infection of PBMCs increases the cell surface expression of several activation/differentiation markers on monocytes with the relative rank order of ICAM-1>CD40>>CD86>CD80>HLA class I-class II (Fig. 7). Kinetics studies revealed that the expression levels of these activation markers increased with time, reaching maximal levels at 5 days postinfection with EBV (data not shown). Levels of CD14, ICAM-2, ICAM-3, CD15, CD23 (Fig. 7), CD16, CD32, and CD64 (data not shown) remained unchanged at 1 day postinfection, indicating that the observed phenotypic changes are specific and not the result of the global up-regulation of activation/differentiation markers. In parallel with the phenotypic and morphological changes, a notable increase in cluster formation of the cells in EBV-infected cultures was observed without leading to plastic adherence of the cells (S. Salek-Ardakani, J. R. Arrand, unpublished observations).



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  		FIGURE 7. EBV induces monocyte maturation to macrophages with phenotypic characteristics of APCs. PBMCs were cultured in the presence or the absence of EBV (0.2 x 105 TFU/ml). After 1 day in culture, cells were harvested and double stained with a PE-Cy5-conjugated anti-CD14 mAb and Abs recognizing specific cell surface markers. Samples were analyzed by flow cytometry. All data were analyzed after gating on viable CD14+ cells. The histograms display the number of CD14+ monocytes at various intensities of fluorescence. Gray histograms, unstained cells; dotted histograms, uninfected cultures; thick-lined histograms, EBV-infected cultures. The experiment was repeated three times. Typical results, from a single experiment, are presented.

 
To assess whether a direct interaction of EBV with monocytes contributes to the phenotypic changes observed using the PBMC system, we tested the effect of EBV on the maturation of purified monocytes. Monocytes were enriched from PBMCs and infected or mock-infected with EBV. EBV infection failed to up-regulate the cell surface expression of HLA class II, CD80, and CD86, and ICAM-1 on purified monocytes (Fig. 8, A–D), implying that EBV induces monocyte maturation indirectly through paracrine secretion of one or more cytokines. Accordingly, transfer of supernatants from EBV-infected, but not mock-infected, cultures to unstimulated monocytes increased the cell surface expression of HLA class II, CD80, and CD86, and ICAM-1 to similar levels as those achieved after EBV infection of PBMCs (Fig. 8, E–H, third row). IFN-{alpha} and IFN-{gamma} are known to be expressed after EBV infection of PBMCs. As these cytokines promote phenotypic changes in monocytes resembling those reported in this study, we reasoned that IFN-{alpha} and IFN-{gamma} may be responsible for the EBV-induced phenotypic changes in monocytes. In concordance, exogenous addition of either recombinant IFN-{alpha} or IFN-{gamma} alone is sufficient to induce modulation of marker expression that is similar or identical with the pattern of markers expressed by monocytes after EBV infection of PBMCs (Fig. 8, E–H). To determine the relative contributions of these cytokines to the EBV-induced phenotypic changes, PBMCs were infected with infectious virus in the presence or the absence of saturating concentrations of neutralizing anti-IFN-{alpha} or anti-IFN-{gamma} Abs for 1 day, after which the expression of each cell surface marker was determined by direct immunofluorescence and analyzed by FACS. Anti-IFN-{gamma}-neutralizing Ab inhibited the EBV-induced increase in the percentage of CD14+/HLA class II+ and CD14+/CD86+ cells within PBMCs by ~65 and 50%, respectively, whereas it had little or no effect on the percentage of CD14+/CD80+ (<31.58%) and CD14+/ICAM-1+ (<12.73%) cells (Fig. 8, compare rows 2 and 5). In contrast, anti-IFN-{alpha}-neutralizing Ab completely inhibited the EBV-induced increase in the percentage of CD14+/CD86+ cells, whereas it had no effect on the percentage of CD14+/HLA class II+ cells. These results suggest that IFN-{alpha} and IFN-{gamma} are required for optimal induction of CD86 and HLA class II cell surface expression on monocytes, respectively. Interestingly, addition of anti-IFN-{alpha}-neutralizing, but not anti-IFN-{gamma}-neutralizing, Abs to EBV-infected PBMCs increased the percentage of CD80+/CD14+ and ICAM-1+/CD14+ cells, indicating that IFN-{alpha} may also have negative regulatory effects on CD80 and ICAM-1 cell surface expression on monocytes. Addition of rIFN-{alpha} to unstimulated PBMCs (Fig. 8, F and H), purified monocytes (Fig. 8, B and D), or monocytes infected with EBV (Fig. 8, B and D), however, markedly enhanced the percentages of both CD80+/CD14+ and ICAM-1+/CD14+ cells. Taken together, these results point to the existence of multiple signals that regulate monocyte maturation and activation after EBV infection of PBMCs.



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  		FIGURE 8. EBV induces monocyte maturation indirectly, through induction of multiple cytokines. A–D, Purified monocytes were cultured in the presence or the absence of EBV (0.2 x 105 TFU/ml), EBV plus IFN-{alpha} (0.2 ng/ml), or IFN-{alpha} (0.2 ng/ml) alone. After 1 day in culture, cells were harvested and double stained with PE-Cy5-conjugated anti-mAbs against the monocyte/macrophage-specific marker, CD14, and mouse Abs recognizing the cell surface markers indicated. E–H, PBMCs were cultured in medium alone, EBV (0.2 x 105 TFU/ml), supernatants from 1-day EBV-infected PBMC (EBV/S/N), EBV plus saturating concentrations (15 µg/ml) of anti-IFN-{alpha}- and anti-IFN-{gamma}-neutralizing Abs, or IFN-{alpha} (2 ng/ml) and IFN-{gamma} (2 ng/ml) alone. One day postinfection with EBV, cells were harvested and double stained as described in A. All data were analyzed after gating on viable CD14+ cells. The x-axis shows the fluorescence intensity (log scale, 4 decades), whereas the y-axis depicts the relative cell number. The percentage of CD14+ cells expressing each cell surface marker under investigation is given in the upper right corner of each histogram. Gray histograms, unstained cells. The data are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Monocytes are among the first cells to encounter microbial pathogens after the initial infection and may represent one of the primary cellular targets for EBV. Recent findings have demonstrated the ability of EBV to infect and to replicate in human monocytes (18) and macrophages (17). Furthermore, cell fractionation experiments suggest an important accessory role for monocytes in B cell transformation (50). To gain a better understanding of the role of monocytes during EBV infection, we examined the effect of EBV on peripheral blood monocytes from multiple donors. We show that EBV inhibits spontaneous apoptosis of monocytes via a mechanism that requires the interaction of the virus with its receptor and is largely dependent on prompt release of IFN-{alpha}. In addition, EBV infection of PBMCs caused a dramatic increase in the size and granularity of the monocytes along with changes to the expression of several cell surface markers indicative of their differentiation to macrophages with phenotypic characteristics of potent APCs.

We elected to use cultured PBMCs so that monocyte-related phenomena dependent on soluble factors released from other cell types could be observed. Indeed, virally induced cytokines proved to be crucial for the effects seen; EBV infection had little effect on the viability of purified monocytes, and the transfer of filtered supernatants from infected PBMCs to uninfected cultures showed that the inhibition of monocyte apoptosis was due to early (within 24 h) release of a soluble factor later identified as IFN-{alpha}. The way in which monocytes respond to EBV appears to be largely cytokine dependent. For example, EBV triggers apoptosis of purified monocytes cultured in the presence of GM-CSF plus IL-4, thereby inhibiting their differentiation into DCs (22). However, this effect is not seen in the absence of the exogenously added cytokines (22). Recent studies have suggested that during antiviral immune responses, exposure of monocytes to IL-4 may not represent the earliest physiological signal for monocyte survival and differentiation and can dramatically alter their natural response to innate cytokines such as IFN-{alpha} (51, 52, 53, 54). The pattern of cytokines produced after EBV infection of PBMCs closely resembles that found during an in vivo infection (in the form of infectious mononucleosis) (26, 27, 28, 29, 30, 31, 32, 33). Thus, we think that the observations made during our experiments may extend to newly recruited monocytes and tissue macrophages present at the site of EBV infection in vivo.

Experiments involving the transfer of supernatants from EBV-infected cultures to naive monocytes and the addition of neutralizing anti-IFN-{alpha} mAbs to EBV-infected PBMCs revealed that paracrine release of IFN-{alpha} is critical for the full monocyte survival-enhancing potential of the virus, and that it is released within 24 h after EBV infection. Although other cytokines, such as GM-CSF, IL-1{beta}, and IFN-{gamma}, are up-regulated as a result of EBV infection of PBMCs and can contribute to the survival of monocytes, the neutralizing Ab experiments suggested that they do not play a role in the inhibition of monocyte apoptosis by EBV. Monocytes produce IFN-{alpha} in response to several viruses, such as HSV-1 and HIV (55, 56); however, this is not the case with respect to EBV. Cell fractionation experiments conducted by Lotz et al. (29) have identified B cells and NK cells as the main source of IFN-{alpha} in EBV-infected PBMCs. Thus, EBV-infected B cells and activated NK cells are likely to be an important source of IFN-{alpha} in our experiments, which would be consistent with our observation that EBV has little effect on the viability of purified monocytes. The precise role of B and NK cell-derived IFN-{alpha} during EBV infection remains to be determined. Addition of exogenous IFN-{alpha} at concentrations similar to those reported to be released after EBV infection of PBMCs (26, 29, 31) inhibited the spontaneous apoptosis of purified monocytes exposed to EBV, implying that B and NK cell-derived IFN-{alpha} can directly enhance monocyte survival in the context of EBV infection. Based on our results described above, we propose that the prompt release of IFN-{alpha} from B and NK cells after EBV infection may represent an early important survival signal responsible for maintaining the number of monocytes and macrophages at the site of viral infection and/or reactivation.

To date, the regulatory effect of IFN-{alpha} with respect to human CD14+ monocyte survival in the context of a virus infection has not been reported. Earlier reports suggested that depending on the type of virus infection and cellular target, IFN-{alpha} can either promote or inhibit the process of apoptosis (57, 58). More recently, IFN-{alpha} was shown to inhibit the spontaneous apoptosis of two subsets of immature DCs, CD11c+ (pDC1 or myeloid DC) and CD11c– (pDC2 or lymphoid DC) isolated from human peripheral blood (59). Interestingly, Kadowaki et al. (60) demonstrated that type 2 DC precursors produce very large quantities of IFN-{alpha} within 24 h in response to HSV infection, which was shown to maintain the survival of the cells during 3 days of culture. Based on this the authors proposed that IFN-{alpha} may act as an autocrine survival factor for some DC precursors. Our study further confirms the antiapoptotic potential of IFN-{alpha}. Furthermore, it provides important new insight into the biological role of IFN-{alpha} in the context of a viral infection by demonstrating that it can critically enhance native human CD14+ monocyte survival in a paracrine manner.

Activation of monocytes leads to their differentiation into either macrophages or DCs and is associated with an increased capacity to present Ags to T cells and to coordinate adaptive immune responses. Our results show that EBV infection of PBMCs induces profound morphological changes in monocytes, readily detected as a time-dependent increase in size (FSC) and granularity (SSC). This is paralleled by a significant and rapid (within 24 h) up-regulation of several cell surface molecules (HLA class I and II, ICAM-1, CD80, CD86, and CD40) that have been implicated as important determinants in Ag-presenting capacity of monocytes/macrophages.

Our results indicate that virally induced soluble factors are responsible for the maturation of monocytes observed in the PBMC system. Accordingly, a neutralizing anti-IFN-{gamma} Ab inhibited the EBV-induced HLA class II and, to a lesser extent, CD86 cell surface expression on monocytes, whereas it had little or no effect on CD80 and ICAM-1 expression. IFN-{alpha}, in contrast, was shown to be absolutely required for CD86, but not HLA class II cell surface expression, implying that multiple cytokines are responsible for specific EBV-induced phenotypic alterations on monocytes. Interestingly, IFN-{alpha} was shown to specifically reduce the cell surface expression of CD80 and ICAM-1 expression on monocytes present within EBV-infected PBMCs, whereas it dramatically enhanced their expression on uninfected PBMCs or purified monocytes exposed to EBV. Thus, the overall outcome of the EBV-induced monocyte activation process probably reflects the balance of positive and negative signals provided by several cytokines secreted by EBV-infected and neighboring EBV-negative inflammatory cells. Additional experiments are required to determine the identity and source of these EBV-induced soluble factors.

In view of the findings described in this report, we hypothesize that the IFN-{alpha}-induced enhancement of monocyte survival and maturation in response to EBV infection may have important physiological relevance. EBV-induced IFN-{alpha} occurs before secretion of detectable cytokines and most likely represents the first line of defense against the infection. Earlier reports have demonstrated that IFN-{alpha} is highly effective in directly inhibiting EBV-induced B cell proliferation and transformation for a very short (24-h) period after infection before the transformed phenotype is established (61). Once transformation has occurred, B cells become insensitive to direct action of IFN-{alpha} and are subject to regulation by cytotoxic T cells (61). Macrophages have been shown to be very efficient in processing EBV and stimulating specific T cell responses that inhibit EBV-induced B cell transformation (10). Our results suggests that the prompt release of IFN-{alpha} after EBV infection may influence the generation of anti-viral T cell-mediated immune responses by modifying their activation through the regulation of monocyte differentiation. This highlights a potential mechanism by which IFN-{alpha} could indirectly exert an additional effect at a later time during the anti-EBV-immune responses. It is also conceivable that IFN-{alpha}-induced enhancement of monocyte survival could contribute to their Ag presentation and T cell stimulation, leading to a boost in the antiviral responses. Similar IFN-{alpha} effects have been observed in response to HIV (52, 57), LCMV (58), HSV (60), and influenza virus (62). Paradoxically, we speculate that a short exposure to IFN-{alpha} may be sufficient to prolong the survival of monocytes long enough for them to provide a transient advantage to the virus, allowing those cells that have internalized the virus or that have absorbed it on their surface to mediate the dissemination of the virus with subsequent infection of B cells during the first critical days after primary infection. Similar mechanisms have been demonstrated for CMV (63), HIV (64), and measles virus (65). Our findings may also have significance for EBV-mediated transformation of B cells. Recently, Wilson et al. (50) provided evidence of an accessory role for monocytes in the transformation of B cells derived from cord blood. They demonstrated that B cells grown in cultures depleted of monocytes were transformed by EBV less efficiently than those grown in the presence of monocytes. The enhanced survival of monocytes by IFN-{alpha} may be critical in prolonging the production of cytokines, such as GM-CSF and IL-6 (produced by these same cells in response to EBV), both of which can dramatically enhance the transformation of B cells by EBV (66, 67). This is an interesting question worthy of further study.

In summary, our results provide new insights into the interaction between EBV and human monocytes and raise the possibility that these cells may serve as a vehicle for the dissemination of the virus as well as being active participants in eliciting EBV-specific CD4+ and CD8+ T cell responses during acute infection. A greater understanding of the interplay among EBV, IFN-{alpha}, and monocytes/macrophages should lead to a new appreciation of their role in both protective and pathogenic aspects of EBV infection with the potential for development of new immunotherapeutic strategies for the treatment of EBV-associated malignancies.


   Acknowledgments
 
We thank Mike Hughes and Jeff Barry for their technical assistance with flow cytometry, Alan Rickinson for the gift of EBV virions, Andrew Morgan for 72A1, and Graham Roberts for rgp350.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Shahram Salek-Ardakani, Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: ssalek{at}liai.org Back

2 Abbreviations used in this paper: DC, dendritic cell; 7-AAD, 7-aminoactinomycin D; FDA, fluorescein diacetate; FSC, forward scatter; SSC, side scatter; TFU, transforming unit. Back

Received for publication December 17, 2003. Accepted for publication April 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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