Regression of a Murine Gammaherpesvirus 68 ... - Journal of Virology

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of S11 lymphocytes does not occur if the cells are implanted into normal .... tumor-infiltrating lymphocytes may serve as a signal for mono- cyte or NK cell ...
JOURNAL OF VIROLOGY, Apr. 2001, p. 3480–3482 0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.7.3480–3482.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 7

Regression of a Murine Gammaherpesvirus 68-Positive B-Cell Lymphoma Mediated by CD4 T Lymphocytes KEVIN A. ROBERTSON, EDWARD J. USHERWOOD,†

AND

ANTHONY A. NASH*

Department of Veterinary Pathology, University of Edinburgh, Edinburgh EH9 1QH, United Kingdom Received 27 October 2000/Accepted 5 January 2001

Murine gammaherpesvirus 68-infected S11 cells were injected subcutaneously into nude mice. Adoptively transferred restimulated lymphocytes consistently elicited the regression of S11 tumors. CD4 T lymphocytes were most effective in preventing tumor formation, and immunohistochemistry highlighted populations of CD4 T cells in regressing tumors. Lymphoproliferative disorders are a common feature of gammaherpesvirus infection. For example, two gammaherpesviruses recognized as infecting humans, Epstein-Barr virus (EBV) and human herpesvirus 8, are associated with a range of lymphocyte malignancies (1, 2, 3). Infection of inbred laboratory mice with murine gammaherpesvirus 68 (MHV-68) results in approximately 9% of these animals developing tumors, and immunohistochemical analyses have revealed that CD4 T cells are consistently found infiltrating these malignancies (8). Several tumor cell lines have been derived from afflicted mice, the most significant of which is termed S11. S11 cells display the characteristics of B lymphocytes and are latently infected with MHV-68. Significantly, if S11 lymphocytes are implanted subcutaneously into nude mice, tumors rapidly develop. Subcutaneous proliferation of S11 lymphocytes does not occur if the cells are implanted into normal, immunocompetent mice. This suggests that T lymphocytes play a role in the responses of mice to this cell line (9). To confirm this hypothesis, the present study was undertaken to investigate whether an immune response to S11 cells could be restored in athymic nude mice by the adoptive transfer of activated, antigen-specific T-lymphocyte subsets into these animals. Splenocytes were obtained from BALB/c mice 25 days after intranasal infection with MHV-68. Cells for restimulation were transferred to 24-well flat-bottom tissue culture plates or upright T75 tissue culture flasks at a concentration of 2 ⫻ 106 cells/ml with 2 ⫻ 105 ␥-irradiated (6000R) S11 cells. These cells were cultured together in RPMI 1640 medium with 10% fetal calf serum, penicillin-streptomycin (final concentrations, 70 and 10 ␮g/ml, respectively), and L-glutamine (2 mM final concentration), and this mixture is referred to as RPMI 1640⫹. Restimulated lymphocytes were harvested after 7 days in culture, washed in RPMI 1640⫹, and counted. Normal splenocytes were obtained from uninfected BALB/c mice. Injections of 107 restimulated, immune or normal spleno-

cytes in sterile phosphate-buffered saline were administered via an intraperitoneal route to female BALB/c (H-2d) nude mice (Harlan UK Limited) in 200-␮l aliquots. The mice were then partially anesthetized, and 2 ⫻ 107 S11 cells were injected subcutaneously into each animal’s flank. Tumor growth was assessed by measurement of the tumor diameter at appropriate times after implantation. Tumor growth profiles are presented in Fig. 1 and 2. In agreement with the previous study of Usherwood et al. (9), S11 cells rapidly proliferated in control nude mice (Fig. 1). Subcutaneous tumors in these animals grew from around 6 mm (⫾1 mm) in diameter at 6 days postimplantation to an average of 12.75 mm (⫾1.75 mm) in diameter after 19 days. In contrast, animals that received S11 restimulated lymphocytes were able to mount an effective immune response to the lymphoma cell line. S11 tumors in this group of mice decreased in size from 5.5 mm in diameter at 6 days postimplantation to less than 1 mm in diameter 19 days after implantation. The nude mice that received splenocytes from infected or uninfected BALB/c mice displayed an intermediate ability to control S11 tumor growth. S11 tumors did not noticeably increase or decrease in size from day 6 onwards in either of these groups. In this instance, the naı¨ve splenocytes were probably mounting a primary response to the S11 graft, whereas the splenocytes from infected mice were responding to viral antigens previously encountered in MHV-68-infected donor mice. Throughout these experiments, S11-restimulated lymphocytes were consistently more effective in inducing tumor regression than lymphocytes taken from mice 25 days after MHV-68 infection. It is likely that this is due to the presence of a higher frequency of tumor-specific lymphocytes in populations of cells subjected to a period of in vitro restimulation with irradiated S11 cells. To assess the role of CD4 and CD8 T-lymphocyte subsets in the regression of S11 tumors in nude mice, groups of animals received complete S11-restimulated splenocytes from infected mice or restimulated splenocytes depleted of CD4 and/or CD8 T lymphocytes. To deplete the T-lymphocyte subsets, spleen cells were incubated with anti-CD4 (YTS191.1) and/or antiCD8 (YTS169.4) antibody both at a final concentration of 100 ␮g/ml along with rabbit complement (Lowtox-H; Cedarlane Laboratories Ltd.) for 1 h at 37°C. Lymphocytes were then washed three times in RPMI 1640⫹ medium. To confirm that

* Corresponding author. Mailing address: Department of Veterinary Pathology, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom. Phone: (44)131 650 6164. Fax: (44)131 650 6511. E-mail: [email protected]. † Present address: Trudeau Institute, Inc., Saranac Lake, NY 12983. 3480

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FIG. 1. Diameter of subcutaneous S11 tumors in nude mice following the adoptive transfer of splenocyte populations. (A) Nude mice were injected intraperitoneally with 107 restimulated, immune, or normal splenocytes. All mice were then injected subcutaneously with 107 S11 cells on the right flank, and tumor development was assessed by measurement of the tumor diameter at the times indicated. Each point represents the mean diameter of four tumors. Error bars represent the standard error of the mean. }, tumor only; ■, tumor plus normal splenocytes; Œ, tumor plus immune splenocytes; ⫻, tumor plus S11 restimulated immune splenocytes. (B) S11 tumors in control animals which received no adoptive transfer of cells. (C) A group of animals which received an adoptive transfer of CD8-depleted lymphocytes at the time of tumor implantation. Both photographs were taken at 19 days post-tumor implantation. White squares highlight the subcutaneous tumors.

lymphocyte depletions were successful, cell populations were analyzed by flow cytometry. Depletion resulted in a greater than 99% reduction in the number of CD4 and/or CD8 lymphocytes present in transferred populations of cells. All mice were then given a subcutaneous injection of S11 cells as was done before. Once again, S11 cells rapidly proliferated in untreated nude mice (Fig. 2). The average tumor in this group of animals increased from around 6 mm in diameter 4 days after implantation to 15.5 mm (⫾0.5 mm) in diameter after 11 days (Fig. 1A and B). In this experiment, no tumor expansion occurred in nude mice which had received splenocytes deficient in CD8 T cells. It is notable that tumors did not reappear in any of these mice up to 100 days after implantation. In contrast, complete restimulated splenocytes were less effective in limiting tumor development. This result indicates that CD8 T cells may actually interfere with the anti-tumor activity of CD4 T cells. Further work is required to verify this hypothesis. Depleting CD4 cells only, or both CD4 and CD8 T lymphocytes from transferred cells, resulted in significant S11 proliferation in nude mice. Tumors in these mice grew to around 13 mm in diameter before the animals were sacrificed. Taken together, these results suggest that CD4 T lymphocytes play a significant role in eliciting tumor regression in this model system. To confirm this finding, the experiment was

FIG. 2. CD4⫹ T cells elicit S11 tumor regression in nude mice. (A) Nude mice were injected intraperitoneally with 107 splenocytes that were either complete, CD4 depleted, CD8 depleted, or CD4 and CD8 depleted. All mice were then injected subcutaneously with 107 S11 cells on the right flank, and tumor development was assessed by measurement of the tumor diameter at the times indicated. Each point represents the mean diameter of four tumors. Error bars represent the standard error of the mean. }, tumor only; 䊐, complete splenocytes; Œ, CD4-depleted splenocytes; E, CD8-depleted splenocytes; ■, no CD4 or CD8 lymphocytes. (B) Tumor section immunostained with a rat anti-mouse CD4 monoclonal antibody. CD4⫹ T cell infiltration (X) was mainly observed in the vicinity of blood vessels (Y) with sporadic staining in other areas of the tumors analyzed. Original magnification, ⫻200. Bar, 0.2 mm. Arrows show positive staining with relevant antibody.

repeated and consistent results were obtained (results not shown). To investigate cellular infiltration into subcutaneous S11 grafts, sections of tumors were stained with antibodies specific for MHV-68, the macrophage marker F4/80, B220, CD4, and CD8. This indicated that macrophages (results not shown) and CD4 T lymphocytes (Fig. 2B) had migrated into tumors. Interestingly, throughout this study, CD8 T-lymphocyte infiltration of S11 tumors was never observed.

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This study demonstrates that T lymphocyte responses to S11 lymphoma cells can be restored in athymic nude mice. In this model, CD4 T lymphocytes efficiently controlled subcutaneous S11 proliferation while the depletion of CD8 T lymphocytes from transferred cell populations had little effect on tumor regression. This contrasts with data obtained from experiments involving the treatment of EBV-associated lymphoproliferative disease (EBV-LPD) in SCID mice in which EBV-specific CD4 T lymphocytes were unable to prevent the development of EBV-LPD (5). In the nude mouse model, CD4 T lymphocytes might be instigating an anti-tumor response in several ways. CD4 T cells may be migrating to tumors and directly destroying S11 lymphoma cells via a major histocompatibility complex class II molecule–T-cell receptor interaction. Against this idea, there is currently no evidence which suggests that CD4 cytotoxic T lymphocytes are generated during the immune responses of mice to MHV-68. Therefore, CD4 T cells could be eliciting an anti-S11 response directly or indirectly via the production of cytokines. Local production of cytokines, such as gamma interferon or granulocyte-macrophage colony-stimulating factor, by restimulated CD4 T lymphocytes may have a direct negative effect on S11 proliferation. However, it seems more likely that these cytokines are initiating an anti-tumor delayed-type hypersensitivity response in the nude mice. Nude mice have significant, functional populations of macrophages and NK cells (10). The secretion of cytokines such as gamma interferon by tumor-infiltrating lymphocytes may serve as a signal for monocyte or NK cell recruitment and activation. It has been suggested that cytokine production by EBVinfected lymphoblastoid cell lines (LCLS) may be responsible for the initiation of nonspecific immune responses to Burkitt’s lymphoma (BL) cell lines in nude mice (10). The fact that S11 cells proliferate in untreated nude mice argues against the possibility of cytokine production by these lymphoma cells being important in initiating nonspecific immune responses. It is unclear why CD8 T lymphocytes alone were incapable of eliciting tumor regression in this model. Evidence suggests that these cells play a significant role in the acute immune responses of mice to MHV-68 infection (7). Further, an MHV68-derived epitope, presented in the context of S11 major histocompatibility complex class I molecules, has now been identified (4). It is possible that CD8 T lymphocytes, when transferred into nude mice, are functionally active and yet are unable to suppress the rapid proliferation of S11 cells in this environment.

J. VIROL.

This model of S11 tumor regression seems to share a number of features with murine models of BL development and immunotherapy. This is in agreement with the findings of Usherwood et al. (9), who proposed that, phenotypically, the S11 line more closely resembles BL lines than LCLs. However, in vitro studies have shown that the S11 line is more immunogenic than BL lines, which express a tightly restricted EBV latent protein profile. In the future, characterization of MHV-68 infection of S11 cells may reveal that this cell line displays characteristics of both LCLs and BL lines. It is hoped that further study in this field will produce information which is applicable to the treatment of human diseases, such as EBVLPD, BL, or even Kaposi’s sarcoma.

Kevin Robertson was supported by a studentship from the Biotechnology and Biological Sciences Research Council, United Kingdom. Thanks to Heather Brooks for expert technical assistance with the immunohistochemistry mentioned in this article. REFERENCES 1. Biberfeld, P., B. Ensoli, M. Sturzl, and T. F. Schulz. 1998. Kaposi sarcomaassociated herpesvirus human herpesvirus 8, cytokines, growth factors and HIV in pathogenesis of Kaposi’s sarcoma. Curr. Opin. Infect. Dis. 11:97–105. 2. Brooks, L. A., A. J. Wilson, and T. Crook. 1997. Kaposi’s sarcoma-associated herpesvirus (KSHV) human herpesvirus 8 (HHV8)—a new human tumour virus. J. Pathol. 182:262–265. 3. Greenblatt, R. M. 1998. Kaposi’s sarcoma and human herpesvirus-8. Infect. Dis. Clin. N. Am. 12:63. 4. Husain, S. M., E. J. Usherwood, H. Dyson, C. Coleclough, M. A. Coppola, D. L. Woodland, M. A. Blackman, J. P. Stewart, and J. T. Sample. 1999. Murine gammaherpesvirus M2 gene is latency-associated and its protein a target for CD8(⫹) T lymphocytes. Proc. Natl. Acad. Sci. USA 96:7508–7513. 5. Lacerda, J. F., M. Ladanyi, D. C. Louie, J. M. Fernandez, E. B. Papadopoulos, and R. J. O’Reilly. 1996. Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced B cell lymphoproliferations in xenografted C.B-17 Scid/Scid mice. J. Exp. Med. 183:1215–1228. 6. Sprent, J. 1974. Migration and lifespan of circulating B lymphocytes of nude (nu/nu) mice, p. 11–22. In J. Rygaard and C. O. Povlsen (ed.), Proceedings of the First International Workshop on Nude Mice, Aarhus. Fischer, Stuttgart, Germany. 7. Stevenson, P. G., and P. C. Doherty. 1998. Kinetic analysis of the specific host response to a murine gammaherpesvirus. J. Virol. 72:943–949. 8. Sunil-Chandra, N. P., J. Arno, J. Fazakerley, and A. A. Nash. 1994. Lymphoproliferative disease In mice infected with murine gammaherpesvirus-68. Am. J. Pathol. 145:818–826. 9. Usherwood, E. J., J. P. Stewart, and A. A. Nash. 1996. Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice. J. Virol. 70:6516–6518. 10. Wolf, J., A. Draube, H. Bohlen, A. Jox, S. Mucke, M. Pawlita, P. Moller, and V. Diehl. 1995. Suppression of Burkitt’s lymphoma tumorigenicity in nude mice by co-inoculation of EBV-immortalized lymphoblastoid cells. Int. J. Cancer 60:527–533.