Generation of Tumor-Specific T Lymphocytes for the Treatment of Posttransplant Lymphoma
Background The incidence of lymphoproliferative disease, including B-cell lymphomas (BCL) in patients who have undergone heart or combined heart-lung transplants, has been reported to be as high as 15%. The majority of these tumors contain Epstein-Barr virus (EBV) DNA and regress when immunosuppressive agents are discontinued. This tumor regression is thought to be secondary to cytotoxic T lymphocytes (CTL) reactive to EBV-infected cells whose function is impaired in patients receiving immunosuppressive agents. We hypothesize that EBV-CTL expanded in the absence of these agents may demonstrate an antitumor effect against an EBV-expressing human BCL in vitro and in vivo.
Methods and Results An EBV-expressing BCL from a heart transplant recipient was isolated and expanded in culture. EBV-CTL were generated by stimulation of peripheral blood leukocytes with irradiated autologous tumor cells in low-dose interleukin-2. Autologous BCL, HLA-mismatched BCL, lymphokine-activated killer target cell line (Daudi), and the natural killer target cell line (K562) were used in a standard 4-hour cytotoxicity assay using 51CrO4 after 7, 14, and 28 days of stimulation. There was significant percent specific lysis of autologous BCL targets (78%) at an effector-to-target ratio as low as 20:1 as compared with control cells. EBV-CTL were then adoptively transferred into SCID mice (provided by Duke University Vivarium) that had been engrafted with autologous BCL 7 days before. There was a significant survival advantage to those mice engrafted with EBV-CTL as compared with control cells.
Conclusions The results indicate that ex vivo expansion of EBV-CTL in the absence of immunosuppressive agents results in a population that has significant antitumor activity. This strategy may be useful in the generation of EBV-CTL that might be effective antitumor agents in transplant recipients with EBV-associated lymphomas.
The incidence of lymphoproliferative disease, including BCLs in patients who have undergone heart or combined heart-lung transplants, has been reported to be as high as 15%. The mortality associated with these tumors that appear more than 1 year posttransplant is approximately 70%. The majority of these BCLs contain EBV DNA as demonstrated by DNA hybridization studies, polymerase chain reaction, and immunohistochemistry.1 2 3
As many as 89% of these tumors regress in thoracic organ transplant patients when immunosuppression is discontinued if the tumor appeared within the first year of transplantation.4 5 6 This phenomenon is thought to be the result of the repopulation of EBV-CTL, which are suppressed by cytotoxic agents such as cyclosporine. Therefore, it is hypothesized that autologous EBV-CTL expanded in the absence of these immunosuppressive agents may demonstrate an antitumor effect against an autologous EBV-expressing human BCL in vitro and in vivo.
Isolation of Posttransplant Lymphoma and Human Cell Lines
After informed consent was obtained a tumor was obtained from a patient who had developed a BCL 7 years after an orthotopic heart transplant for ischemic cardiomyopathy. The tumor was minced and passaged. At surgery, portions of excised tumor were placed in either sterile saline, snap-frozen in liquid nitrogen, or fixed in neutral-buffered formalin. Those portions in saline were mechanically dispersed into single-cell suspensions and layered and placed in culture in 25-cm2 flasks (CoStar) with RPMI 1640 (Sigma Chemical Co)/20% FCS with supplemental antibiotics. BJAB is an ATCC (American Type Culture Collection, Rockville, Md) line isolated from human Burkitt’s lymphoma that is EBV negative.
FACS was performed by single- or double-staining of isolated cells by direct techniques using phycoerythrin- or fluorescein isothiocyanate–conjugated monoclonal antibodies. Cells were washed with cold PBS containing 0.5% BSA and 0.1% NaN3. The cells were then incubated with directly labeled antibodies for 30 minutes at 4°C. The cells were washed and analyzed in an EPICS C flow cytometer (Coulter) at 488 nm for percent positivity on a log fluorescent scale. Background control tubes were incubated with directly labeled class-matched mouse Igs. Monoclonal antibodies CD4 (OKT4), CD8 (OKT8), and CD19 (OKB-PanB) from Ortho Diagnostics and CD3 (Leu-4), CD19 (Leu-12), CD20 (Leu-16), CD22 (Leu-14), CD23 (Leu-20), and CD56 (Leu-19) from Becton-Dickinson were used for single and dual color-flow cytometry.
SCID mice were obtained from a hysterectomy-rederived central inbred colony of defined flora-gnotobiotic stock maintained at Duke University. After transfer to a biosafety level 3 isolation facility, mice were maintained in filter-capped Micro-Isolator cages (Lab Products Inc). Cages were housed within a HEPA-filtered Blickman isolator system (Blickman), and mice were fed sterilized rodent chow. Before all manipulations, Micro-Isolator cages were transferred to a laminar flow Bioguard hood (The Baker Co), and animals were handled aseptically by investigators wearing sterile gloves, masks, and gowns. Blood samples were obtained from all mice before their induction into experiments by retroorbital sinus bleeding. Serum from mice was screened for murine Ig by ELISA and total murine Ig levels were quantified using goat antimouse Ig. Mice with Ig levels greater than 0.01 mg/mL, indicative of leakiness of the SCID defect, were excluded from study.
The primary human tumor and SCID mice tumors were processed simultaneously. Formalin-fixed specimens were dehydrated and embedded in paraffin, and 5-μm sections were cut and stained with hematoxylin and eosin for morphological evaluation.
Frozen sections (5 μm) prepared in the usual fashion were stained by an avidin-biotin procedure with a panel of monoclonal antibodies including CD20 (L26, B cell antigen; Dako), CD3 (pan-T cell antigen; Becton-Dickinson), and CD45 RO (UCHL-1, pan leukocyte antigen; Dako).
Frozen sections were also stained with antibodies to EBNA 2 (EBV-encoded nuclear antigen 2) and LMP-1 (EBV-encoded latent membrane protein) from Dako Corp.
Both frozen and paraffin sections were stained with polyclonal rabbit antihuman to kappa and lambda Ig light chain (Calbiochem-Behring Corp) for determination of clonality.
In Situ Hybridization
A biotinylated probe for EBV-DNA (Epstein-Barr virus Bioprobe-labeled probe; Enzo Diagnostics) was used for the detection of EBV genomes in formalin-fixed sections.
Generation of Anti–EBV CTL
Peripheral blood from the patient was diluted 1:3 with PBS and layered over a Ficoll-Hypaque gradient. The mononuclear cell layer was then washed twice and cocultivated with gamma-irradiated BCL in AIM-V media with 5% heat-inactivated autologous plasma for 7 days at a concentration of 1×106 cell/mL with a responder-to-stimulator ratio of 5:1. Repeated stimulations of EBV-CTL were performed at days 7 and 21 with irradiated BCL and a similar responder-to-stimulator ratio. After the second stimulation, recombinant interleukin-2 (rIL-2); 10 IU/mL, Chiron) was added, and the cells were counted and expanded every 3 days with the addition of fresh rIL-2.
Target cells were incubated with 51CrO4 washed, resuspended at 5×104 cells/mL, and combined in triplicate wells of 98-well round-bottom plates with effector cells for E-T (effector to target) ratios of varying numbers. Final volumes of each well were adjusted to 0.2 mL with AIM-V media. Wells containing only culture media and target cells or 5% triton and target cells served as spontaneous and maximum 51Cr release controls, respectively. The plates were incubated at 37°C in 5% CO2 for 4 hours; then 0.10 mL of medium from each well was removed for counting in a Packard Prias gamma spectrometer. Percent specific lysis was calculated by standard methods.
Tumor Cell Engraftment and Development
A reliable and reproducible model of human B-cell tumors (SCID-BCL) in SCID mice has been developed in this laboratory. After engraftment with PBL (peripheral blood leukocytes), engraftment with PBL followed by inoculation with EBV, and engraftment of LCL (lymphoblastoid cell line) results in the development of B cell tumors. To test the effect of adoptive transfer of EBV-CTL on SCID/BCL development 25×106 EBV-CTL were inoculated into the peritoneal cavity of SCID mice 7 days after IP inoculation of 5×106 tumor cells or Dulbecco’s PBS (Gibco). Mice were monitored twice weekly, weight and examination for tumor development. Mice were anesthetized with halothane (Fluothane; Ayerst), killed by cardiac puncture, and autopsied. At autopsy, thoracic and abdominal cavities were examined, and tumors were sectioned and cryopreserved in liquid nitrogen or fixed in 10% neutral-buffered formalin. All procedures were approved and conducted in accordance with institutional guidelines.
The patient’s primary tumor and those that developed in the SCID mice were polyclonal B-cell type as shown in Fig 1⇓. The presence of EBV was confirmed in the primary patient’s tumor and the SCID mice by in situ hybridization as seen in Fig 2⇓. FACS analysis revealed significant staining of the tumor cells with B-cell markers CD 19 (95%) and 20 (84%) as well as the EBV receptor CD21 (92%). The EBV viral products EBNA 2 and LMP were noted by immunohistochemistry.
As shown in the Table⇓, the EBV-CTL generated progressed to 97% CD3+ (pan-T cell) and 88% CD8+ (cytotoxic T cell) after 28 days of stimulation. The majority of CD8+ cells were positive for S6F1 (cytotoxic T cell). The population of B cells or natural killer (NK) target cells diminished with time, to near zero.
Effective cytolysis of the autologous targets was demonstrated by EBV-CTL 7, 14, and 21 days after stimulation as seen in Fig 3⇓. No cytolysis of HLA-mismatched EBV-expressing cell lines was seen. Minimal cytolysis of the NK target K562 was seen on day 7, which diminished by day 14. There was no activity against LAK (lymphokine activated killer) cell target Daudi (data not shown).
Adoptive Transfer of EBV-CTL
Results of SCID-BCL and BJAB-BL engraftment and survival as well as the inoculation of EBV-CTL are shown in Fig 4⇓. Engraftment of posttransplant B-cell lymphoma and Burkitt’s lymphoma led to the characteristic tumor development and death of the animals in ≈50 days. Animals inoculated with EBV-CTL 7 days after engraftment with the EBV-negative cell line BJAB (Burkitt’s lymphoma) showed no significant difference in survival from the animals engrafted with tumor and then given PBS (P=NS via log rank test). In contrast, animals given autologous EBV-CTL 7 days after engraftment of EBV-expressing BCL had no deaths (P<.05 via log rank test).
Cell-mediated immune responses to EBV infection are considered to be more crucial to the outcome of infection than the humoral responses. A lymphocytosis is composed primarily of CD3+/CD8+ T lymphocytes and is usually seen in acute EBV infection. This early response appears to be a polyclonal T cell activation. Two to 8 weeks after active EBV infection, a more EBV-specific T-cell response can be seen by in vitro proliferation to viral antigen and EBV-specific HLA-restricted lysis of EBV-infected B cells.7 8 9 EBV-CTLs have been cloned from the blood of EBV seropositive donors and inhibit cytotoxicity that is highly specific for EBV-infected cells and are HLA restricted.10 11
Wherever productively infected cells are cleared by the immune system, latently infected B cells persist for the life of the host. In fact, B cells infected with EBV become immortalized B-cell lines and provide a model system of EBV latency.12 13
Evidence suggests that cellular immune response may be effective in controlling EBV-associated LPDs and BCLs in patients who have undergone transplantation.14 15 The term LPD applies to the development of continuously proliferating B lymphocytes, presumably stimulated by EBV infection. This is believed to be related to the failure of the immune system, especially the T-cell population, which is suppressed by drugs such as cyclosporine, to respond normally to EBV-infected B lymphocytes.16 17 The fact that the risk of LPD and the mortality from it increases with the duration of immunosuppression adds validity to this theory. In approximately 89% of patients in whom BCL developed within 1 year of transplantation, there was complete regression of the lesions after reduction of immunosuppressive therapy alone.18 19 Thus, the decrease in number or function of EBV-CTL may allow for unchecked proliferation of EBV-driven LPD.
One report on EBV-associated LPD after allogeneic bone marrow transplantation documented successful eradication of disease after infusions of donor-derived pooled leukocytes. Because of the known high-frequency of EBV-specific CTL precursors in the blood of seropositive normal donors, it is reasonable to suggest that donor-derived EBV-reactive T cells had a critical role in the response noted. More specific therapy with just EBV-CTL may provide a tailored approach with less systemic side effects. Continued study of the mechanisms underlying the cause of EBV-driven LPD and the cellular immune response, including the EBV-specific CTL response to it, may provide novel treatment strategies against BCLs in the organ-transplant recipient.
Selected Abbreviations and Acronyms
|CTL||=||cytotoxic T lymphocytes|
|EBV-CTL||=||CTLs specific against EBV|
|FACS||=||flow cytometric analysis|
|SCID||=||severe combined immunodeficient mice|
The secretarial assistance of Maureen Coyle is gratefully acknowledged.
Reprint requests to J. Michael DiMaio, MD, Department of Surgery, PO Box 3050, Duke University Medical Center, Durham, NC 27710.
- Copyright © 1995 by American Heart Association
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