Nakamine,* Samuel J. Pirruccello,* Jack R. Davis,* KirkW. Beisel ..... Davey DD, Kamat D, Laszewski M, Goeken JA, Kemp JD,. Trigg ME, Purtilo DT, Davis J, ...
AmericanJournal ofPathology, Vol. 137, No. 3, September 1990 Copyright © American Association ofPathologists
Rapid Communication Characterization of Epstein-Barr Virus-induced Lymphoproliferation Derived from Human Peripheral Blood Mononuclear Cells Transferred to Severe Combined Immunodeficient Mice Motohiko Okano,* Yuichi Taguchi,* Hirokazu Nakamine,* Samuel J. Pirruccello,* Jack R. Davis,* Kirk W. Beisel,* Kimberly L. Kleveland,* Warren G. Sanger,*#f Renee R. Fordyce,t David T. Purtilo*ti" From the Department of Pathology, atnd Microbiology,* the Ceniterfor Humani Genetics,t the Department of Pediatrics,* and the Eppley Institute for Research in Cancer andAllied Diseases,11 University ofNebraska Medical Center, Omaha, Nebraska
Mice with severe combined immunodeficiency (SCID) received 6 X 107 fresh human peripheral blood mononuclear cells (PBMC) intraperitoneally from Epstein-Barr virus (EBV)-seropositive and -seronegative donors. B95-8 EBV was inoculated intraperitoneally and intravenously to the mice 6 weeks after transfer of seronegative PBMC. Three offour mice transferred with PBMCfrom two EBV-seropositive donors and two offour micefrom two EBV-seronegative donors inoculated with EBV developed fatal EBV-induced lymphoproliferative disease within 6 to 10 weeks. These tumors were oligoclonal orpolyclonal by cytoplasmic immunoglobulin expression. Furthermore no consistent clonal chromosomal abnormalities were shown. Cell lines established from these tumors showed low cloning efficiency in soft agarose. In addition, latent membrane protein, B-lymphocyte activation antigen (CD23), and cell-adhesion molecules (ICAM- 1, CD 18) all were expressed in the EBV-positive infiltrating lymphoproliferative lesions in each mouse. These results suggest that lymphoid tumors are comparable to lymphoblastoid cell lines immortalized by EBV and are not malignant lym-
phomas such as Burkitt's lymphoma. This model may be useful for investigating mechanisms responsiblefor the growing numbers of lymphoproliferative diseases that are occurring in patients with inherited or acquired immunodeficiencies. (Am J Pathol 1990, 137:517-522)
Epstein-Barr virus (EBV) is a ubiquitous virus that subclinically infects during infancy and early childhood.1 However it causes infectious mononucleosis and is etiologically associated with Burkitt's lymphoma (BL) and undifferentiated nasopharyngeal carcinoma.1 These diseases are postulated to occur mainly due to host genetic and/or environmental factors that often govern immunosurveillance. Furthermore, in patients with inherited or acquired immunodeficiencies, EBV is implicated etiologically in growing numbers of lymphoproliferative diseases.1 Whether these lymphoproliferative diseases are malignant by classical criteria is obscured by the findings of variable clonality by immunohistochemical staining for immunoglobulins, Southern blot hybridization analysis using the heavy-chain J-region (JH) fragment of the immunoglobulin gene complex, and by the spontaneous regression of the lesions when immunosuppressive drugs are withdrawn after transplantation.24 In addition, EBV-induced lymphoproliferative lesions in patients with immunodeficiency express viral and cellular gene products, including latent membrane protein (LMP), EBV-determined Supported in part by PHS CA30196 and CA36727, awarded by the National Cancer Institute, DHHS, the State of Nebraska Department of Health, LB506, and the Lymphoproliferative Research Fund. Accepted for publication June 25, 1990. Address reprint requests to Motohiko Okano, MD, PhD, Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 South 42nd St., Omaha, NE 68193-3135.
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nuclear antigen (EBNA)-2, B-lymphocyte activation marker (CD23), and cell-adhesion molecules.5 In contrast, these antigens generally are down regulated in BL cells in tissue.6 Recently EBV-positive B-cell lymphomas were reported in severe combined immunodeficient (SCID) mice engrafted with human peripheral blood mononuclear cells (PBMC) from EBV-seropositive donors.7 Cannon et a18 reported that the tumors, derived mainly from EBV-seronegative PBMC-transferred SCID mice inoculated by exogenous EBV, showed normal diploid cells without chromosomal abnormalities, oligoclonal or polyclonal proliferations, and high levels of ICAM-1 and LFA-3 expressions. However, depending on the degree of infiltration of EBV-infected cells, interpretation of related antigens may be difficult in this SCID mouse xenograft model. We intended to clarify further the exact status of the lesions regarding mainly malignant potential and EBV-induced activation markers using methods of cloning efficiency and double staining for EBNA and several phenotypic markers.
Materials and Methods C.B.17 SCID/SCID mice (aged 6 to 12 weeks from McLaughlin Research Institute, Great Falls, MT), which lack functional T and B cells,9 were injected with 6 X 107 human PBMC intraperitoneally. Donors included one EBV-seropositive healthy individual who is a carrier of xlinked lymphoproliferative disease (XLP),10 one seronegative healthy individual, one seropositive patient, and one seronegative patient with XLP. Patients with this disease are exquisitely vulnerable to EBV-induced lymphoproliferative diseases.10 Supernatants of cultured B95-8 cells11 containing 1 06 immortalizing units of EBV were inoculated intravenously (0.2 ml) and intraperitoneally (0.8 ml) 6 weeks after injection of 6 X 107 PBMC in mice previously engrafted with EBV-seronegative PBMC. The lesions were evaluated histopathologically using hematoxylin and eosin staining. After mincing tissues, mononuclear cells from the lesions were obtained by density-gradient centrifugation using Lymphoprep@R (Nycomed As, Oslo, Norway) and smeared on microscope slides (Baxter Healthcare Corporation, McGaw Park, IL). These preparations and touch imprints of the lesions were fixed with acetone-methanol (1:1, -20°C for 2 minutes). Then they were incubated with EBV-seropositive (EBNA titer = 1:640) and seronegative (EBNA titer < 1:5) reference sera with the complement at 37°C for 40 minutes. Thereafter the preparations were washed with phosphatebuffered saline. After air drying, these cells were incubated with fluorescein-isothiocyanate (FITC)-conjugated goat anti-human C3 (Jackson lmmunoResearch Laboratories, Inc., West Grove, PA) at 37°C for 40 minutes.1 For
double staining, after an additional washing with PBS, they were incubated with rhodamine isothiocyanate-conjugated goat anti-human immunoglobulins specific for different heavy and light chains (Organon Teknika Corporation, Durham, NC) at 370C for 40 minutes. All preparations were viewed with a Nikon Labophot immunofluorescence microscope (Nikon, Inc., Garden City, NY). Furthermore, after EBNA staining, each preparation was incubated with different monoclonal antibodies; anti-LMP (S-12, a gift from D. A. Thorley-Lawson, Tufts University, Boston, MA), anti-ICAM-1 and anti-CD18 (a gift from M. Patarryoyo, Karolinska Institute, Stockholm, Sweden), and anti-CD23 (Coulter Immunology, Hialeah, FL) at 37°C for 40 minutes. After washing with PBS, the preparations were incubated with rhodamine isothiocyanate-conjugated goat anti-mouse IgG (Jackson lmmunoResearch Laboratories) at 370C for 40 minutes. Then they were viewed with an immunofluorescence microscope, as described above. In addition, these cells also were incubated with only each FITC-conjugated goat anti-human C3, rhodamine isothiocyanate-conjugated goat anti-human immunoglobulins, or goat anti-mouse IgG to evaluate the specific staining and nonspecific background. No remarkable nonspecific background was shown. Karyotype analysis was done by standard banding techniques.12 Cells recovered from each lesion also were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 20% heat-inactivated fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), penicillin (100 U/ml), and streptomycin (100 ,tg/ml) to establish lymphoid cell line spontaneously. To evaluate the cell lines for their malignant potential, anchor-independent growth in a semi-solid agar suspension culture was pursued using 1 X 103 cells that were suspended in 2 ml of medium containing 0.3% agarose (DIFCO Laboratories, Detroit, MI).2 Cells were plated in 60-mm polystyrene tissue culture dishes (Corning Glass Works, Corning, NY) containing 0.4% agarose. Cultures were incubated at 370C in a humidified atmosphere of 5% CO2 and examined with a Nikon bright-field microscope (Nikon) after 3 weeks to assess colony formation. Raji cells (BL cells, American Type Culture Collection, Bethesda, MD) were used as a control in this assay. The animal experiments were approved by the Animal Review Committee of the University of Nebraska Medical Center. All blood was obtained after informed consent.
Results Three of four SCID-human chimeric mice engrafted from two EBV-seropositive donors and two of four mice from two EBV-seronegative donors, following exogenous inoc-
ulation of EBV, developed systemic lymphoproliferative disease within 6 to 10 weeks after injection of PBMC. The histopathologic evaluation of hematoxylin-eosin-stained
EBV-induced Lymphoproliferation in SCID-human Mice 519 AJP Septenmber 1990, Vol. 13 7, No. 3
Figure 1. A: Photoinicrograph of a lynmpho-
prolije'rative lesioni in a kidney of SCID niouse (no. 1-1) shouwingfratures of immunoblastic liompphoma. Note the displace-
ment of the niormal tissuie struictuires anid massive niecrosis (N). IIematoxi'lin and eosini staiilinlg; magiiitcatioii is X 75. B: The same lesioni at higher magnificationi (X480). The large atipical immunoblastic cells shou' a fiirl' monomorphic morphology. C: Epstein -Barr virus-determined niiuclear anztigeni (EBNA) staininlg of cells fromo the samee lesioni (mlagiiitcationi, X400).
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Table 1. Properties ofEBV-iniduced Ljmphoproliferative Diseases Derivedfrom Human Peripheral Blood Mononuclear Cells Transfevrred into Severe Combined ImmunodeficientMice Direct examination of lesion
Mouse no. 1-1
EBV status of donor* Positive
-2 2-1 -2
Negative***
3-1
Positive
-2 4-1 -2
Positive Negative***
Organ Liver Spleen Kidney Lymph nodel PW¶ PBL** No obvious tumor formation Spleen No obvious tumor formation Liver
Spleen PW\1 Liver Spleen PWT Thymus No obvious tumor formation
% Colony-
% EBNApositive cells
Immunoglobulin expressiont
10.5 2.1 8.1 25.1 30.1 NT
KX/.ya KXuya KXIyTa NT
Yes Yes Yes Yes Yes Yes
