1 Laboratory of Transplant Immunology and Pediatric Hematology/Oncology, IRCCS Policlinico S. Matteo, Pavia; 2 Department of Clinical and Experimental Medicine, Second University of Napoli; 3 Divisione di Oncologia Medica Falck, Department of Oncology, Ospedale Niguarda Ca Granda, Milano; 4 Transplant Immunology Unit, S. Martino Hospital, Genova, Italy
Received 23 July 2003; revised 4 September 2003; accepted 17 September 2003
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ABSTRACT |
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The outcome of patients with nasopharyngeal carcinoma (NPC) presenting as advanced-stage disease or failing conventional radio-chemotherapy is poor. Thus, additional forms of effective, low-toxicity treatment are warranted to improve NPC prognosis. Since NPC is almost universally associated with EpsteinBarr virus (EBV), cellular immunotherapy with EBV-specific cytotoxic T lymphocytes (CTLs) may prove a successful treatment strategy.
Patient and methods
A patient with relapsed NPC, refractory to conventional treatments, received salvage adoptive immunotherapy with EBV-specific CTLs reactivated ex vivo from a human leukocyte antigen-identical sibling. EBV-specific immunity, as well as T-cell repertoire in the tumor, before and after immunotherapy, was evaluated.
Results:
CTL transfer was well tolerated, and a temporary stabilization of disease was obtained. Moreover, notwithstanding the short in-vivo duration of allogeneic CTLs, immunotherapy induced a marked increase of endogenous tumor-infiltrating CD8+ T lymphocytes, and a long-term increase of latent membrane protein 2-specific immunity.
Conclusions:
Preliminary data obtained in this patient indicate that EBV-specific CTLs are safe, may exert specific killing of NPC tumor cells in vitro, and induce antitumor effect in vivo.
Key words: cellular immunotherapy, cytotoxic T lymphocytes, EpsteinBarr virus, latent membrane protein 2, nasopharyngeal carcinoma
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Introduction |
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NPC is almost universally associated with EpsteinBarr virus (EBV), which is present in tumor cells in a latency II state, characterized by the expression of a restricted number of viral proteins, with latent membrane protein (LMP)1 and LMP2 as the only immunogenic antigens [3]. Recent work has shown that NPC cells are capable of immunological processing for cytotoxic T-lymphocyte (CTL) recognition [4, 5]. Based on these data, and on the encouraging results obtained by cellular immunotherapy with EBV-specific CTL lines in the prevention and treatment of the latency III, EBV-related post-transplant lymphoproliferative disease [6, 7], pilot trials of cellular immunotherapy for other EBV-associated cancers have been conducted [8, 9].
However, so far there is no evidence that EBV-specific CTLs reactivated from NPC patients are capable of lysing autologous tumor cells in vitro and mediating tumor killing in vivo. An increase in EBV-specific CTL precursor (CTLp) frequency was observed after infusion of autologous virus-specific CTLs in four patients with NPC, but no data on in vitro anti-tumor activity were available, and no clinical benefit could be observed [9].
We describe the case of a NPC patient who received EBV-specific CTLs reactivated from a human leukocyte antigen (HLA)-matched family donor, which showed in vitro cytotoxicity towards the patients tumor cells, and, once transferred in vivo, induced temporary stabilization of disease and enhancement of the patients EBVLMP2-specific T-cell responses.
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Study design |
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Approval for this study was obtained from the Institutional Review Board, and both donor and patient gave written informed consent before enrolment.
EBV-specific CTL expansion and administration
EBV-specific CTLs were reactivated and expanded in vitro according to a method previously reported, following good laboratory practice standard procedures [7]. In brief, EBV-specific CTLs were prepared from fresh or frozen peripheral blood mononuclear cells (PBMCs), plated in 2 ml X-VIVO 20 medium (BioWhittaker, Walkersville, MD, USA) with 2% autologous plasma, at 2 x 106 cells per well and stimulated with irradiated autologous B-lymphoblastoid cell line (LCL) at a responder-to-stimulator (R:S) ratio of 40:1. After 10 days, cultures were restimulated with irradiated autologous B-LCL at a R:S ratio of 4:1. Starting on day 14, 20 U/ml recombinant interleukin-2 (rIL-2; Hoffman- La Roche, Basel, Switzerland) were added to the wells, and the cultures were subsequently restimulated weekly with irradiated autologous B-LCL in the presence of rIL-2.
Before cryopreservation, T cells were examined for immunophenotype, sterility and EBV specificity in a standard 51Cr-release assay against a panel of targets including donor B-LCL, recipient EBER+ tumor cells, and recipient phytohemagglutinin (PHA) blasts. Tumor cells were obtained from a baseline tumor biopsy, after seeding tumor sections overnight in a culture flask in a small volume of IMDM (Gibco, Grand Island, NY, USA) supplemented with 20% fetal calf serum (FCS; Sigma, St Louis, MO, USA). Adherent cells were maintained with weekly passages in IMDMFCS medium, until a substantial outgrowth of cells was observed, and then analyzed for immunophenotype and EBER expression. Early-passage neoplastic cells were used to minimize the possibility of loosing primary characteristics as a consequence of extensive in vitro re-culturing.
CTL infusion schedule included a first dose of 2 x 107 CTL/m2, and 3-weekly subsequent doses of 4 x 107 CTL/m2.
Analysis of CTL presence and persistence at the tumor site
Presence and persistence of infused CTLs of donor origin was monitored through T-cell receptor repertoire analysis by CDR3 spectratyping. In detail, CDR3 size length analysis was employed to characterize T-cell clonotypes on donor EBV-specific CTLs, lymphocytes derived from tumor site tissue before and after cell therapy, and on the peripheral blood of patients after CTL infusion, according to a previously described method [10].
Evaluation of specific immunity
EBV- and LMP2-specific immunity before and after immunotherapy was evaluated by limiting dilution analysis of CTLp [7], by measuring the frequency of IFN-secreting cells in a ELISPOT assay [11], and by reactivation of specific CTLs.
In detail, to evaluate CTLp frequency to EBV, responder cells were stimulated with autologous irradiated B-LCL in a limiting dilution assay. Briefly, 24 replicates of decreasing numbers (8 x 104, 6 x 104, 4 x 104, 2 x 104, 104, 5 x 103, 2.5 x 103) of cryopreserved PBMCs, used as responder cells, were seeded in 96-well round-bottom microplates in a final volume of 200 µl RPMI (Gibco)FCS medium in the presence of 104 autologous irradiated (7000 rads) B-LCL. On days 7 and 14, the cultures were restimulated with 104 autologous irradiated B-LCL. On day 21, the plates were split and tested against autologous or HLA-mismatched B-LCL in a standard cytotoxicity assay. For ELISPOT assay, 96-well multiscreen filter plates (MAIPS 4510; Millipore, Bedford, MA, USA) were coated with 100 µl of primary antibody (IFN; Mabtech, Nacka, Sweden) at 2.5 µg/ml, and incubated overnight at 4°C. PBMCs were thawed and cultured overnight in RPMIFCS medium before use in the assay, and were then seeded in the absence or in the presence of peripheral blood monocyte-derived dendritic cells (DC) pulsed with vaccinia-LMP2. Controls included wells plated with vaccinia-ßgal-pulsed DC. After incubation for 24 h at 37°C, 100 µl of biotinylated secondary antibody (Mabtech; 0.5 µg/ml) was added, and plates were then processed according to standard procedure. IFN
-producing spots were counted using a Elispot reader (Bioline, Torino, Italy). The number of spots per well was calculated after subtraction of assay background, quantitated as the average of 24 wells containing only sterile complete medium, and specific background, quantitated as the sum of cytokine spots associated with responders alone, and responders plated with vaccinia-ßgal-pulsed DC.
Tumor-infiltrating lymphocytes were obtained by culturing phosphate-buffered saline-washed tumor sections in RPMIFCS medium containing IL-2 (100 IU/ml). Medium was replaced on day +6, and on day +10 cells were harvested, phenotyped and used fresh or cryopreserved.
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Results and discussion |
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Tumor biopsies were performed before and 2 weeks after completion of CTL infusion schedule. Immunohistochemical staining showed a marked increase of CD8+ lymphocytes in the tumor after CTL therapy (data not shown). We proceeded to analyze the T-cell repertoire, and found that a marked increase of the message for T-cell repertoire was present after the CTL infusion (Figure 2). Moreover, the repertoire was found to be identical, as for CDR3 length, before and after CTL infusion, but different to the infused CTLs (Figure 2B). To better characterize these T cells, we sequenced some of the BV families found to be more expanded. The results concerning BV18 (Figure 2C) show the same prominent peak before and after infusion, and a 3bp shorter peak in the CTLs. The sequencing data revealed that the same clone was found before and after infusion, and no sequence motif was found in the CDR 3 region of the same family in the CTLs, suggesting that donor CTLs were not present at the time of the analysis.
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Preliminary data obtained in this patient indicate that EBV-specific CTLs are able to exert specific killing of patients NPC cells in vitro, can be used safely, and may show antitumor effect in vivo. Given the short in vivo duration of allogeneic CTLs, immunotherapy of NPC may be implemented by the use of autologous EBV-specific CTLs [13, 14]. Recently, it has been reported that immunization with LMP2 peptide-pulsed autologous DCs boosted epitope-specific T-cell responses in NPC patients, inducing partial clinical responses in selected patients [15]. Thus, to improve antitumor response, LMP2 and/or LMP1-specific CTLs [16, 17] could be employed, supported by low-dose IL-2 administration [12].
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Acknowledgements |
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FOOTNOTES |
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