Affiliations of authors: Laboratory of Immunovirology, Department of Microbiology and Immunology, and Pediatric Research Center, University of Montreal and Sainte-Justine Hospital, Montreal, PQ, Canada.
Correspondence to: José Menezes, Ph.D., D.V.M., Laboratory of Immunovirology, Sainte-Justine Hospital 3175, Côte-Ste-Catherine Rd., Montreal, PQ, Canada H3T 1C5 (e-mail: jmenezes{at}justine.umontreal.ca).
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ABSTRACT |
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INTRODUCTION |
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Primary infection with EBV leading to IM is associated with a remarkable immune stimulation that results in the activation and expansion of lymphocytes with cytotoxic and suppressor effector functions (16,17). Although there is a strong humoral response to EBV, it is the cellular immune response that is believed to be primarily responsible for controlling EBV infection and EBV-transformed cells (18). Different effector mechanisms appear to be involved that might be regulated by cytokines released from activated cells, as well as from accessory cells after their interaction with the virus (19). It is noteworthy that it has been shown that peripheral blood lymphocytes from individuals with EBV-induced IM also display a remarkable major histocompatibility complex (MHC)-unrestricted cytotoxic activity during the acute phase of the disease (20,21).
The relative role of different cytokine-induced cytotoxic effectors in the immune control of tumor cells is not yet clear. In this context, the role of different cytokines in mediating the immune control of EBV-immortalized cells is also unknown. This is particularly true for interleukin 15 (IL-15), which can enhance the activity of cytotoxic lymphocytes. IL-15 was originally isolated from the supernatants of the simian kidney epithelial cell line CV-1/EBNA in 1994 (22). Despite a lack of amino acid sequence homology with interleukin 2 (IL-2), IL-15 has similar tertiary structure and shares many of the biologic activities with this cytokine. These shared activities stem from the common use of the and
chains of the IL-2 receptor (IL-2R) by IL-15 and IL-2 for binding and inducing signal transduction (23). It has been shown that IL-15 is a T-cell chemoattractant (24) and that it can inhibit apoptosis in activated T and B cells (25). In addition, IL-15 acts as a costimulator with IL-12 to facilitate the production of interferon gamma and tumor necrosis factor-
from natural killer (NK) cells (26) and promotes the induction of cytolytic effector cells, including cytotoxic T cells and lymphokine-activated killer cells (22). Although IL-15 shares biologic activities with IL-2, there are several properties of IL-15 that are distinct from those of IL-2. IL-15 uses a distinct
chain (IL-15R
) other than the
chain of the IL-2R (27). In addition, while IL-2 is selectively expressed in activated T cells, IL-15 messenger RNA has been found constitutively expressed in several human tissues (22). This difference in the expression pattern between IL-2 and IL-15 suggests varied in vivo roles for each of these cytokines. Recently, it was shown that IL-15 drives the expansion of CD8+ memory T cells in vivo, while this effect is counterbalanced by IL-2 (28).
Previous reports from our laboratory (2931) have demonstrated that exposure of human peripheral blood mononuclear cells (PBMCs) to different, unrelated viruses including EBV results in immediate enhancement of IL-15 gene expression and in IL-15 secretion by infected PBMCs. More important, all of these studies demonstrated the ability of secreted IL-15 to enhance the cytocidal activity of NK cells immediately after viral infection, thus suggesting an important role for this cytokine in antiviral innate immune response and elimination of virus-infected cells. It was, therefore, of interest to determine whether IL-15 can also generate cytotoxic effectors capable of controlling EBV-transformed/immortalized cells.
Here, we provide evidence demonstrating that the presence of recombinant human IL-15 in EBV-infected human PMBC cultures indeed results in complete elimination of EBV-immortalized cells by IL-15-activated NK and NK-T effectors. The results of this study can be highly pertinent with regard to new immunotherapeutic approaches, particularly against EBV-associated lymphoproliferative disease and possibly also against different virus-associated tumors.
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MATERIALS AND METHODS |
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For investigating the effect of IL-15 on the immune control of EBV-transformed cells, we collected peripheral blood from eight healthy donors, seven of whom were EBV seropositive and one of whom was EBV seronegative. Blood samples were obtained from each donor after giving informed written consent and following approval of this research by this research center's Ethics Committee. The EBV-seropositive and EBV-seronegative status of these donors was defined by the presence or absence, respectively, of antibodies to EBV capsid antigen and to EBNA by use of immunofluorescence assay (see below). PBMCs were isolated by centrifugation (400g at 20 °C for 25 minutes) of blood on a FicollHypaque (Pharmacia Amersham, Biotech AB, Uppsala, Sweden) gradient by the standard procedure (29). Cells were resuspended at a concentration of 1 x 106 cells/mL in complete medium composed of RPMI-1640 medium supplemented with 20% heat-inactivated fetal bovine serum (FBS) containing 100 IU/mL penicillin, 20 µg/mL streptomycin, 1.0 µg/mL gentamycin, and 1.0% glutamine. B cells were recovered from PBMCs by negative immunoselection by use of a commercial kit (Stem Cell Purification System; Stem Cell Technologies, Vancouver, BC, Canada), according to the manufacturer's instructions. Briefly, PBMCs (5 x 107/mL) were incubated for 30 minutes on ice with the antibody cocktail (100 µL/mL) for the enrichment of human B cells, after which the magnetic colloid (60 µL/mL) was added for an additional 30 minutes. Cells were then loaded into purification column, and the purified cells were collected in the run through the column. A similar procedure was used to isolate NK cells [CD8(-)CD56(+)], NK-T cells [CD8(+)CD56(+)], and CD8+ T cells [CD8(+)CD56(-)] from IL-15-treated PBMCs. The purity of the cell populations thus obtained was greater than 95% as determined by flow cytometry analyses.
Cell lines and reagents.
The B958, K562, BJA-B, and EBV-immortalized lymphoblastoid cell line (LCL) cell lines used were cultured at 37 °C in the presence of 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and antibiotics as described previously (32). Human recombinant IL-15 and monoclonal antibodies (MAbs) to human IL-15 (M110 and M112) were a gift from the Immunex Corporation (Seattle, WA). IL-15 was used at a concentration of 50 ng/mL, and anti-IL-15 was used at a concentration of 10 µg/mL throughout this study. These concentrations were determined on the basis of our previous studies (2931). The effectiveness of IL-15 neutralization by anti-IL-15 MAb was evaluated by its ability to inhibit IL-15-induced NK cell activity as described previously (29,31). Anti-LMP-1 MAb (S12 MAb) was provided by E. Kieff (Harvard University, Boston, MA).
Preparation of EBV, infection of cells, and cytokine treatment.
EBV was obtained from cell-free supernatants of EBV-producing B958 cell line as described previously (33) but without the addition of phorbol myristate acetate. The viral preparation used had a titer of 105 EBNA-inducing units/mL as determined by use of BJA-B cells as described previously (33). In preliminary experiments, the addition of 100 µL of this viral preparation (i.e., 104 EBNA-inducing units) to PBMC cultures (106 cells/tube) obtained from different blood samples invariably resulted in the growth of EBV-immortalized cells, which was confirmed by established criteria (33). For EBV infection and cytokine treatment, PBMCs (1 x 106 cells) were incubated with 104 EBNA-inducing units of the viral suspension or mock infected (i.e., with virus-free supernatant) for 60 minutes at 37 °C in 5% CO2. After extensive washing with complete medium, EBV- and mock-infected cells were resuspended in 1 mL of complete medium in the presence or absence of IL-15 (50 ng/mL), anti-IL-15 (10 µg/mL), or IL-15 plus anti-IL-15. The cells were then cultured in microplate wells (2 x 105 cells per 200 µL/well) and were refed twice weekly with complete medium supplemented as above. We monitored the growth of EBV-transformed cells with clearly visible cell clusters by microscopy, as well as by the expression of EBV-encoded LMP-1 and EBNA complex by immunoblotting and immunofluorescence techniques, respectively.
Cell proliferation assay.
We measured proliferation 72 hours after the indicated treatment by adding 1 µCi of [3H]thymidine 6 hours before the cells were harvested. Cells were then harvested on glass-fiber filter paper, and the incorporation of radioactivity was determined by use of a liquid scintillation counter.
Cell depletion.
PBMCs prepared as described above were incubated with anti-CD3 or anti-CD16 MAbs for 60 minutes on ice. Cells were then washed twice and treated with rabbit complement for 60 minutes at 37 °C. The cell depletion procedure was repeated (twice) to maximize cell purity. The efficacy of depletion was assessed by fluorescence-activated cell sorter analysis of viable cells and was found to be greater than 95%.
Cell cytotoxicity assay.
Cytotoxicity assay was performed by use of a standard 51Cr-release assay as described previously (31). Each experiment was carried out in triplicate, and the results are presented as the mean ± 95% confidence intervals (CIs) of three independent determinations.
Western blot analyses.
Western blot analyses were performed by use of a standard procedure (29). S12 MAb (1 : 2000) specific for LMP-1 protein and goat anti-mouse immunoglobulin G coupled to horseradish peroxidase were used in immunoblotting assay. The immunoreactive bands were detected by use of enhanced chemiluminescence reagents (Du Pont NEN, Boston, MA) on x-ray film.
Indirect immunofluorescence.
EBNA detection was carried out by use of the anticomplement immunofluorescence test as described by Reedman and Klein (34). Stained preparations were examined under a fluorescence microscope.
Statistical analysis.
The results from triplicate wells of all in vitro cell proliferation assays or from quadruplicate wells of all in vitro cell lysis analyses were averaged and are reported as mean ± 95% CIs. The 95% CI was calculated as 1.96/
n, where
= the standard deviation of data and n = the number of tests. Differences were analyzed for significance by Student's t test, and a P value of less than .05 was considered to be statistically significant.
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RESULTS |
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For the investigation of the effect of IL-15 on the growth of EBV-immortalized cells in vitro, PBMCs from seven EBV-seropositive individuals and one EBV-seronegative healthy individual were infected with the use of the lymphocyte-transforming EBV strain B958 and incubated in the presence or absence of IL-15 and/or anti-IL-15 antibodies for 21 days. Microscopic analyses showed that, while PBMCs that were incubated without viral infection for 21 days did not show any sign of EBV-induced transformation (Fig. 1, A), EBV infection of PBMCs in vitro resulted in EBV-induced transformation with clearly visible cell clusters (Fig. 1, B
). On the other hand, incubation of EBV-infected PBMCs in the presence of 50 ng/mL of IL-15 resulted in the inhibition of EBV-induced transformation and visible cell clusters through 21 days after infection (Fig. 1, C
). Simultaneous addition of IL-15 and a MAb specific to IL-15 (10 µg/mL) to EBV-infected PBMC cultures resulted in the development of clusters of EBV-transformed cells (Fig. 1, D
), thus illustrating the specificity of IL-15's effect in the control of the growth of EBV-infected/transformed cells.
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LMP-1 is an EBV-encoded, cell-transforming protein expressed in EBV-immortalized lymphocytes (15). Immunoblotting analysis was carried out to determine LMP-1 protein expression in cultures from EBV-seronegative and EBV-seropositive donors 21 days after infection in the presence or absence of IL-15. As shown in Fig. 2 (lanes 16) , LMP-1 protein was detected in EBV-infected cells grown in the absence of IL-15 (lane 4) but was undetectable when EBV-infected cells were cultured in the presence of IL-15 for 21 days (lane 5). Moreover, addition of anti-IL-15 antibodies to EBV-infected PBMCs cultured in the presence of IL-15 resulted in the detection of LMP-1 (lane 6). No LMP-1 band was detected from PBMCs cultured without any (i.e., virus or IL-15) treatment (lane 3). The EBV-negative cell line K562 (lane 1) and EBV-positive cell line LCL (lane 2) were used as negative and positive controls, respectively. Taken together, these data clearly demonstrate that the presence of IL-15 resulted in the elimination of EBV-transformed cells in PBMC cultures.
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To determine the effect of IL-15 treatment on EBV-transformed cells, we added IL-15 to cultures at later stages of infection after evidence of EBV-induced transformation. PBMCs from an EBV-seronegative individual were either infected with EBV or mock infected, as described in the "Materials and Methods" section. Two weeks later, when EBV-induced transformation of B lymphocytes was confirmed by microscopic observation of cellular growth and EBNA antigen expression by immunofluorescence (i.e., with >60% EBNA-positive cells in EBV-infected cultures), cells were cultured in the presence or absence of IL-15 for an additional 10-day period. The effect of IL-15 on the growth of EBV-transformed cells in these cultures was then determined by detecting EBNA expression. As shown in Fig. 3, the presence of IL-15 for this additional 10-day period resulted in the elimination of EBV-infected PBMC cultures. In contradistinction, EBV-infected PBMCs incubated in the absence of IL-15 showed 93% or more EBNA positivity when cultured for a similar period of time. Mock-infected PBMCs cultured in the presence or absence of IL-15 showed 0% EBNA positivity after 25 days of incubation. These results clearly show that IL-15 is effective in controlling the growth of EBV-immortalized cells, whether added initially at the time of viral infection or after viral transformation.
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To determine if IL-15 has any direct inhibitory effect on EBNA synthesis, we cultured cells of the EBV-producer B958 cell line, which contain multiple copies of the EBV genome, for 10 days in the presence or absence of IL-15. Immunofluorescence analysis indicated no substantial changes in the percentage of EBNA-expressing cells between the two culture conditions (83.0% [95% CI = 79.0% to 86.9%] versus 90.2% [95% CI = 83.4% to 97.1%]). Similar results were obtained with a cell line derived after lymphocyte transformation by EBV (LCL) in vitro (91.8% [95% CI = 85.8% to 97.7%] versus 86.2% [95% CI = 81.5% to 91.0%]). In addition, the presence of IL-15 had no notable effect on the viability and growth of B958 cells and LCL, as was determined by trypan blue dye exclusion and cell proliferation assays, respectively (data not shown).
Since B lymphocytes are the targets for EBV-induced immortalization in PBMC cultures (33), we further examined whether IL-15 exerts a direct inhibitory effect on the proliferation of freshly isolated B cells infected with EBV. B cells were purified from freshly isolated PBMC from EBV-seronegative and EBV-seropositive individuals by use of negative selection procedure as described in the "Materials and Methods" section. Purified B cells were then infected with EBV, cultured in the presence or absence of IL-15 and/or anti-IL-15 antibodies, and examined microscopically for the appearance of EBV-induced transformed cell clusters. Microscopic examination and EBNA expression 15 days after infection (data not shown) confirmed the transformed status of EBV-infected B cells in these cultures. IL-15 treatment for 15 days did not inhibit cell proliferation of purified EBV-infected/transformed B lymphocytes as compared with the cells cultured without IL-15 (Fig. 4). In fact, IL-15 stimulated the proliferation of EBV-infected B lymphocytes (Fig. 4
). The addition of a MAb to IL-15 abrogated the proliferative effect of IL-15 treatment on B cells (P = .004). Taken together, these results suggest that IL-15 has no direct inhibitory effect on EBV-infected/transformed B cells; rather it has a stimulatory effect on their proliferation.
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To learn about the nature of the effector cells involved in the observed inhibition of EBV-transformed cells in PBMC cultures in the presence of IL-15, we first investigated the role of the effector populations (T and NK cells) on the IL-15-mediated effect. PBMCs were depleted of T cells (CD3+) or NK cells (CD16+) and were cultured after EBV infection in the presence or absence of IL-15 or IL-15 plus anti-IL-15 MAb. EBV-induced immortalization was assessed in these cultures by microscopic observation and monitoring of the presence of EBNA-expressing cells as determined by immunofluorescence staining at different time points (see below). As was consistently observed, IL-15 treatment of unfractionated, EBV-infected PBMCs resulted in the complete elimination of EBV-transformed cells as compared with EBV-infected PBMCs without IL-15 treatment (Table 1).
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Effect of IL-15-Induced NK-T Cells on Growth of EBV-Infected/Transformed Cells
The data that we described above clearly indicated that both NK and T cells were involved in the IL-15-induced inhibitory activity against EBV-transformed/immortalized cells. Previous studies (24,31) have shown that IL-15 is a potent activator of both NK and T cells. During the present experiments, we also found that IL-15 treatment of PBMCs resulted in a statistically significant induction of lymphocyte proliferation that was completely abrogated when we used IL-15 neutralized with anti-IL-15 antibody (Fig. 5, A) (P = .0001). Therefore, we thought it important to determine the phenotype as well as the cytolytic activity of the proliferating cells. For this purpose, IL-15-treated PBMCs were first analyzed by flow cytometry by use of different fluorescein isothiocyanate-conjugated MAbs against NK- and T-cell surface markers. Data from flow cytometry analyses revealed that, while IL-15 had a notable proliferative effect on CD8+ T cells (Fig. 5, B
), no such proliferative effect was observed on CD4+ T lymphocytes (Fig. 5, C
) as compared with untreated cells. Furthermore, two-color flow cytometry analysis of IL-15-treated PBMCs clearly showed that, while IL-15 specifically and considerably induced proliferation of CD8(+)CD56(+) NK-T-cell population (Fig. 6, A
), it did not induce CD3()CD16(+) NK cell proliferation (Fig. 6, B
). For the direct determination of the cytolytic capacity of these CD8(+)CD56(+) NK-T lymphocytes, this cell population was purified from IL-15-treated PBMCs by negative selection as described in the "Materials and Methods" section. Purified NK-T lymphocytes were then incubated with target cells and tested for their ability to lyse these cells. Our results clearly reveal that these IL-15-activated, purified CD8(+)CD56(+) NK-T lymphocytes are powerful killers at different effector-to-target ratios (Fig. 7, A
). Two other populations, including CD8(+)CD56() T cells and CD8()CD56(+) NK cells were also purified from similar treatment and tested for their cytolytic activity. The data obtained revealed that CD8() CD56(+) NK cells exhibited relatively low cytolytic activity against K562 target cells (at effector-to-target ratio of 40 : 1) (Fig. 7, B
). This cytolytic activity of the NK cells after 21 days of IL-15 treatment did not differ substantially from that observed 24 hours after IL-15 treatment (28.5% [95% CI = 26.6% to 30.4%] versus 33.9% [95% CI = 31.7% to 35.8%], respectively). Furthermore, the number of CD8()CD56(+) cells obtained from IL-15-treated PBMCs was considerably lower than that of CD8(+)CD56(+) NK-T cells isolated from these PBMCs (0.5 x 106 versus 20 x 106, respectively). On the other hand, purified CD8(+)CD56() T lymphocytes from IL-15-treated PBMCs had no remarkable killing activity against K562 target cells (Fig. 7, B
) as compared with both CD8()CD56(+) NK or CD8(+)CD56(+) NK-T cells. Taken together, these results clearly indicate that IL-15 treatment not only sustained the NK cytolytic activity but also induced the expansion of highly cytolytic CD8(+)CD56(+) NK-T lymphocytes.
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DISCUSSION |
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NK and nonspecific T cells were suggested to play an essential role in host defenses aimed at controlling viral spread during the acute phase of infection through the induction of death of virus-infected cells (17). Considering that the abrogation of EBV-transformed/immortalized cells was not due to the direct effect of IL-15 on these cells, we sought to determine the effector cells involved in the elimination of the EBV-infected targets in PBMC cultures. Infection of unfractionated PBMCs with EBV resulted in remarkable growth of immortalized cells after 18 days of infection. However, the growth of EBV-transformed cells was abrogated completely when EBV-infected PBMCs were cultured in the presence of IL-15. Depletion of CD16-positive NK cells clearly revealed the important role of these cytolytic cells immediately after viral infection, since their absence in PBMCs before EBV infection led to accelerated growth of virus-immortalized cells. This is consistent with recently published reports (37,38), in which IL-15 was shown to reduce herpesvirus infection in vitro through the activation of NK cells in short-term experiments. This result together with our present results, showing an incomplete control of EBV-infected/transformed cells at the early stages after infection, strongly indicates that additional effector cells may be required for a complete IL-15-mediated control of cells infected by EBV (and possibly also by other herpesviruses).
Results from the addition of IL-15 to PBMC cultures strongly suggested the ability of IL-15 to exert a stastically significant proliferative effect in cell cultures (P<.001). Phenotypic analysis revealed that IL-15, while having no remarkable effect on the growth of CD4-bearing population, induced the expansion of CD8(+)CD56(+) NK-T lymphocytes during 21 days of culture. Results of cytotoxic assays strongly suggest that the expansion of these highly cytolytic NK-T effector cells in the presence of IL-15 could represent the second step in the induction process of antiviral effector mechanisms by this cytokine, thus ensuring an appropriate and extended innate immune response. Thus, these mechanisms would involve, first, an enhancement of NK cytolytic activity in the early stage of infection and, second, an induction of highly cytotoxic CD8(+)CD56(+) NK-T cells in the later stage of infection. Such NK-T cells have been shown previously to be potent and MHC-unrestricted killer cells that induce cell death in their targets mainly through granule exocytosis (39). Furthermore, IL-15-treated PBMCs also contained a very small number of CD8(-)CD56(+) NK cells that maintained their basal level cytolytic activity in the presence of IL-15 during the incubation time. This result is in agreement with previously published reports in which a picomolar amount of IL-15 was demonstrated to sustain the survival of resting human NK cells for several days (40). In any event, although the precise mechanism of the NK and NK-T-cell effects leading to deletion of EBV-transformed cells is not clear, there is no reason to believe that it is not due to their cytolytic activity against these EBV-positive cells. Further studies will be required to address this issue.
Several lines of evidence validate the important role of IL-15 in the maintenance and expansion of NK-T cells in vivo. Genetic evidence from IL-15-/- mice strongly supports the crucial role of IL-15 in the development of NK-T cells (41). IL-15-/- mice displayed a pronounced reduction in the number of thymic and peripheral NK-T cells as well as a reduced number of splenic memory CD8+ T cells, whereas conventional T-cell numbers were not affected. More important, these mice had an increase in the severity of vaccinia virus-induced pathogenesis compared with control littermates, strongly suggesting a potential association between severity of infection and absence of NK-T and CD8+ T cells (41). Moreover, two IL-15 transgenic mouse models were generated, one expressing the normal variant of the IL-15 gene and the other expressing an alternatively spliced isoform of IL-15 that encoded for the intracellular production of the protein but had impaired secretion. In contrast to the normal IL-15 transgenic mice, mice expressing the spliced isoform of IL-15 showed a substantial decrease in the NK-T cells population. This defect in NK-T cells was highly associated with a lack of resistance to Salmonella infection (42). Of interest, the number of NK-T cells was substantially increased in the peritoneal cavity of mice expressing wild-type IL-15 after Salmonella infection, further emphasizing the role of IL-15 in primary host immune response (42). In addition, data from mice lacking the IL-15R revealed a dramatic reduction in the number of NK-T cells. More important, although IL-2-/- mice had a normal number of NK-T cells, mice deficient in IL-2R
, a component of both IL-15 and IL-2 receptor complexes, showed a critical decrease in the number of NK-T cells (43). Taken together, these data demonstrate a unique and distinct role for IL-15 (as compared with IL-2) in the maintenance and activation of NK-T cells. The importance of IL-15 in immune response as well as in T-cell homeostasis has also been emphasized by a recent study (28) on its key role in the survival and expansion of memory CD8+ T cells.
In conclusion, the present in vitro data clearly show that IL-15 can play a crucial role in the control of EBV-transformed/immortalized cells and that both NK and NK-T lymphocytes are important effectors in this control: NK cells in the early phase, i.e., immediately after EBV infection, and NK-T lymphocytes in later stages after infection, i.e., from about the second to the third week after infection. If these in vitro results are to reflect the human host's antiviral effector mechanisms against EBV-infected/transformed cells in vivo, one might then be tempted to consider the possibility that IL-15-mediated immunotherapy may be of help in patients with EBV-induced lymphoproliferative states and malignancies as well as in patients with chronic active EBV infection. Of interest, IL-15 use seems to present advantages over the use of IL-2, which has been shown to pose a major toxicity problem causing a vascular leak syndrome that can ultimately lead to organ failure when administered in immunotherapy (44). Indeed, when IL-15 and IL-2 were used at a comparable dose in a mouse model of metastatic tumor, IL-15 was found to cause a threefold less pulmonary vascular leak than IL-2 (24). More important, IL-15's unique role in the control of virus-infected/transformed cells through the expansion of a cytotoxic NK-T-cell population would constitute an incentive for experimenting with the use of this cytokine in immunotherapeutic approaches, in particular for the treatment of EBV-induced lymphoproliferative diseases. The fact that IL-15 also contributes to the expansion of memory CD8+ T cells is also highly pertinent in this regard. Furthermore, whether IL-15-induced cytotoxic effectors can play a protective role in patients with chronic active EBV (and other viral) infections remains to be addressed.
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NOTES |
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Present address: E. Sharif-Askari, Department of Microbiology and Immunology, McGill University, Montreal, PQ, Canada.
Present address: L. M. Fawaz, Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, PQ, Canada.
Supported by grants from the Medical Research Council of Canada and the J.-L. Lévesque Foundation (to J. Menezes).
We thank the Immunex Corporation (Seattle, WA) for recombinant human interleukin 15 (IL-15) and anti-IL-15 antibodies, Drs. Caroline Alfieri and Krishna Peri for advice and help during this study, Dr. Yves Théoret for assistance in photography, Dr. Heydar Sadeghi for help with statistical analysis, and Ms. Micheline Patenaude for secretarial assistance (all from Sainte-Justine Hospital, Montreal, Canada). We also thank Dr. Lena Al-Harthi for revising the manuscript (Rush-Presbyterian-St. Lukes Medical Center, Chicago, IL).
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Roizman B, Carmichael LE, Deinhardt F, de-The G, Nahmias AJ, Plowright W, et al. Herpesviridae. Definition, provisional nomenclature, and taxonomy. The Herpesvirus Study Group, the International Committee on Taxonomy of Viruses. Intervirology 1981;16:20117.[Medline]
2 Henle G, Henle W, Diehl V. Relation of Burkitt's tumor-associated herpes-type virus to infectious mononucleosis. Proc Natl Acad Sci U S A 1968;59:94101.[Medline]
3 Neri A, Barriga F, Inghirami G, Knowles DM, Neequaye J, Magrath IT, et al. Epstein-Barr virus infection precedes clonal expansion in Burkitt's and acquired immunodeficiency syndrome-associated lymphoma. Blood 1991;77:10925.[Abstract]
4 Pallesen G, Hamilton-Dutoit SJ, Zhou X. The association of Epstein-Barr virus (EBV) with T cell lymphoproliferations and Hodgkin's disease: two new developments in the EBV field. Adv Cancer Res 1993;62:179239.[Medline]
5
Straus SE, Cohen JI, Tosato G, Meier J. NIH conference. Epstein-Barr virus infections: biology, pathogenesis, and management. Ann Intern Med 1993;118:4558.
6 Young LS, Dawson CW, Clark D, Rupani H, Busson P, Tursz T, et al. Epstein-Barr virus gene expression in nasopharyngeal carcinoma. J Gen Virol 1988;69(Pt 5):105165.[Abstract]
7 Su IJ, Hsieh HC, Lin KH, Uen WC, Kao CL, Chen CJ, et al. Aggressive peripheral T-cell lymphomas containing Epstein-Barr viral DNA: a clinicopathologic and molecular analysis. Blood 1991;77:799808.[Abstract]
8 Chan LC, Srivastava G, Pittaluga S, Kwong YL, Liu HW, Yuen HL. Detection of clonal Epstein-Barr virus in malignant proliferation of peripheral blood CD3+ CD8+ T cells. Leukemia 1992;6:9526.[Medline]
9 Wen S, Mizugaki Y, Shinozaki F, Takada K. Epstein-Barr virus (EBV) infection in salivary gland tumors: lytic EBV infection in nonmalignant epithelial cells surrounded by EBV-positive T-lymphoma cells. Virology 1997;227:4847.[Medline]
10 Knowles DM. Molecular pathology of acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma. Semin Diagn Pathol 1997;14:6782.[Medline]
11 Khan G, Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA. Is EBV persistence in vivo a model for B cell homeostasis? Immunity 1996;5:1739.[Medline]
12 Middleton T, Gahn TA, Martin JM, Sugden B. Immortalizing genes of Epstein-Barr virus. Adv Virus Res 1991;40:1955.[Medline]
13 Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA. Identification of the site of Epstein-Barr virus persistence in vivo as a resting B cell. J Virol 1997;71:488291.[Abstract]
14 Wang F, Gregory C, Sample C, Rowe M, Liebowitz D, Murray R, et al. Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J Virol 1990;64:230918.[Medline]
15
Kieff E. Epstein-Barr virusincreasing evidence of a link to carcinoma. N Engl J Med 1995;333:7246.
16 Sheldon PJ, Hemsted EH, Papamichail M, Holborow EJ. Thymic origin of atypical lymphoid cells in infectious mononucleosis. Lancet 1973;1:11535.[Medline]
17
Tomkinson BE, Wagner DK, Nelson DL, Sullivan JL. Activated lymphocytes during acute Epstein-Barr virus infection. J Immunol 1987;139:38027.
18 Rickinson AB, Moss DJ. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol 1997;15:40531.[Medline]
19 Khanna R, Burrows SR, Moss DJ. Immune regulation in Epstein-Barr virus-associated diseases. Microbiol Rev 201995;59:387405.
20
Seeley J, Svedmyr E, Weiland O, Klein G, Moller E, Eriksson E, et al. Epstein Barr virus selective T cells in infectious mononucleosis are not restricted to HLA-A and B antigens. J Immunol 1981;127:293300.
21 Patel PC, Dorval G, Menezes J. Cytotoxic effector cells from infectious mononucleosis patients in the acute phase do not specifically kill Epstein-Barr virus genome-carrying lymphoid cell lines. Infect Immun 1982;38:2519.[Medline]
22 Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 1994;264:9658.[Medline]
23 Giri JG, Ahdieh M, Eisenman J, Shanebeck K, Grabstein K, Kumaki S, et al. Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J 1994;13:282230.[Abstract]
24 Wilkinson PC, Liew FY. Chemoattraction of human blood T lymphocytes by interleukin-15. J Exp Med 1995;181:12559.[Abstract]
25 Bulfone-Paus S, Ungureanu D, Pohl T, Lindner G, Paus R, Ruckert R, et al. Interleukin-15 protects from lethal apoptosis in vivo. Nat Med 1997;3:11248.[Medline]
26 Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, et al. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 1994;180:1395403.[Abstract]
27 Giri JG, Kumaki S, Ahdieh M, Friend DJ, Loomis A, Shanebeck K, et al. Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. EMBO J 1995;14:365463.[Abstract]
28
Ku CC, Murakami M, Sakamoto A, Kappler J, Marrack P. Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science 2000;288:6758.
29
Flamand L, Stefanescu I, Menezes J. Human herpesvirus-6 enhances natural killer cell cytotoxicity via IL-15. J Clin Invest 1996;97:137381.
30 Atedzoe BN, Ahmad A, Menezes J. Enhancement of natural killer cell cytotoxicity by the human herpesvirus-7 via IL-15 induction. J Immunol 1997;159:496672.[Abstract]
31
Fawaz LM, Sharif-Askari E, Menezes J. Up-regulation of NK cytotoxic activity via IL-15 induction by different viruses: a comparative study. J Immunol 1999;163:447380.
32 Khyatti M, Patel PC, Stefanescu I, Menezes J. Epstein-Barr virus (EBV) glycoprotein gp350 expressed on transfected cells resistant to natural killer cell activity serves as a target antigen for EBV-specific antibody-dependent cellular cytotoxicity. J Virol 1991;65:9961001.[Medline]
33 Menezes J, Jondal M, Leibold W, Dorval G. Epstein-Barr virus interactions with human lymphocyte subpopulations: virus adsorption, kinetics of expression of Epstein-Barr virus-associated nuclear antigen, and lymphocyte transformation. Infect Immun 1976;13:30310.[Medline]
34 Reedman BM, Klein G. Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer 1973;11:499520.[Medline]
35
Armitage RJ, Macduff BM, Eisenman J, Paxton R, Grabstein KH. IL-15 has stimulatory activity for the induction of B cell proliferation and differentiation. J Immunol 1995;154:48390.
36
Trentin L, Cerutti A, Zambello R, Sancretta R, Tassinari C, Facco M, et al. Interleukin-15 promotes the growth of leukemic cells of patients with B-cell chronic lymphoproliferative disorders. Blood 1996;87:332735.
37
Gosselin J, TomoIu A, Gallo RC, Flamand L. Interleukin-15 as an activator of natural killer cell-mediated antiviral response. Blood 1999;94:42109.
38
Ahmad A, Sharif-Askari E, Fawaz L, Menezes J. Innate immune response of the human host to exposure with herpes simplex virus type 1: in vitro control of the virus infection by enhanced natural killer activity via interleukin-15 induction. J Virol 2000;74:7196203.
39
Mehta BA, Schmidt-Wolf IG, Weissman IL, Negrin RS. Two pathways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+ CD56+ killer cells. Blood 1995;86:34939.
40
Carson WE, Fehniger TA, Haldar S, Eckhert K, Lindemann MJ, Lai CF, et al. A potential role for interleukin-15 in the regulation of human natural killer cell survival. J Clin Invest 1997;99:93743.
41
Kennedy MK, Glaccum M, Brown SN, Butz EA, Viney JL, Embers M, et al. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 2000;191:77180.
42
Nishimura H, Yajima T, Naiki Y, Tsunobuchi H, Umemura M, Itano K, et al. Differential roles of interleukin 15 mRNA isoforms generated by alternative splicing in immune responses in vivo. J Exp Med 2000;191:15770.
43 Ohteki T, Ho S, Suzuki H, Mak TW, Ohashi PS. Role for IL-15/IL-15 receptor beta-chain in natural killer 1.1+ T cell receptor-alpha beta+ cell development. J Immunol 201997;159:59315.
44 Lotze MT. Future directions for recombinant interleukin-2 in cancer: a chronic inflammatory disorder. Cancer J Sci Am 1997;3 Suppl 1:S1068.[Medline]
Manuscript received April 30, 2001; revised August 29, 2001; accepted September 17, 2001.
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