Ex Vivo Priming of Naïve T Cells Into EBV-Specific Th1/Tc1 Effector Cells by Mature Autologous DC Loaded with Apoptotic/Necrotic LCL

Abstract

Posttransplant lymphoproliferative disorders (PTLDs) represent life-threatening complications of bone marrow and solid organ transplantation (SOTx). These are B-cell malignancies triggered by Epstein-Barr Virus (EBV) infection in chronically immunosuppressedRestoration of protective anti-Epstein-Barr Virus (anti-EBV) T-cell immunity by adoptive transfer of ex vivo expanded EBV-specific cytotoxic T lymphocytes (CTLs) has been shown to be a safe and effective approach to prevent or treat posttransplant lymphoproliferative disorders (PTLDs) in bone marrow transplant recipients (1-3). Standard published protocols emphasize the effectiveness of using autologous lymphoblastoid cell lines (LCLs) as stimulator cells in boosting memory immune responses to EBV in vitro (2,3). However this same approach has not proven effective in priming naïve anti-EBV T cells in vitro (4,5). As EBV − SOTx recipients are at high risk of developing PTLD, we have been interested in establishing protocols to generate EBV-specific CTLs from these individuals. Studies performed by other groups, and in our laboratory, have recently established the key role of using dendritic cells (DCs) as antigen-presenting cells in activating naïve T cells against EBV epitopes (6,7). In the current study we wanted to assess and compare the ability of DCs loaded with necrotic/apoptotic LCLs in cross-priming autologous naïve EBV-reactive T cells vs. boosting memory T cells, and to further characterize the quality of the resulting immune responses.The CTL lines were evaluated by flow cytometry analyses with MHC Class I/peptide tetramers and mAbs to detect memory/activation markers, ELISPOT analysis for IFN-c production, and in cytotoxicity tests. Results indicate that in our system, in vitro cross-priming promotes both activated poly-epitope viral-specific CD8+ CTLs and Th1-type CD4+ T-cell responses in EBV − donors. The polarized Type 1 pattern of cytokine expression induced by DCs fed with apoptotic/necrotic LCLs correlated with the ability of T cells to mediate cytotoxicity against autologous LCLs, which was further enhanced by addition of rhIL-12 in the first round of in vitro stimulation. These results support the prospective development of DC-based cellular immunotherapy for EBV − SOTx patients at high risk of developing PTLD or patients with refractory PTLD. Materials and Methods Human subjectsSeven immunocompetent adult volunteers were recruited in this study following informed consent under IRB-approved protocol. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood (8), while sera served as material for determination of EBV status. 1369

Processing of Two Latent Membrane Protein 1 MHC Class I Epitopes Requires Tripeptidyl Peptidase II Involvement

“…A possible explanation can be crosspresentation of LMP1 by DCs, because this mechanism has already been proven in a model in which DCs loaded with apoptotic/ necrotic LCLs prime EBV-specific T cells (48,49).…”

Successful In Vitro Priming of EBV-Specific CD8+ T Cells Endowed with Strong Cytotoxic Function from T Cells of EBV-Seronegative Children

“…The standard method, successfully employed to reactivate EBV-specific CTL from virus-seropositive individuals, fails to efficiently expand EBV-specific, HLA class I-restricted CD8+ CTL with high cytotoxic capacity from virus-seronegative individuals (4,16,18). Recently, two studies have demonstrated successful generation of EBV-specific CTL lines displaying CD8+ T-cell-mediated cytotoxicity from adult EBV-seronegative subjects, either by the standard protocol or by stimulation with DC loaded with apoptotic/necrotic LCL (18)(19). These findings were either attributed to the presence of environmental antigenspecific CTL precursors cross-reacting with EBV antigens or to failure to mount a serological response upon previous EBV exposure (18).…”

“…These cells, which likely play a central role in inhibiting proliferation of newly EBV-infected B cells (20), and might contribute help to the CD8+ CTL response, have not been proven to control established PTLD in vivo. Therefore, we elected to CD8+ CTL to Treat PTLD in EBV-Naïve Children (19,30). However, the specific cytotoxicity exerted by those CTL, reactivated from EBV-seronegative adults, was of small magnitude.…”

“…In detail, PBMC were plated in 2 mL X-VIVO 20 medium (BioWhittaker, Walkersville, MD, USA) with 2% autologous plasma, at 2 × 10 6 cells per well and stimulated with irradiated autologous LCL at a responder-to-stimulator (R:S) ratio of 40:1; (II) 2 × 10 6 responder PBMC per well were co-cultured with DC pulsed with apoptotic autologous LCL at a R:S ratio of 20:1. Apoptosis was obtained by c irradiation (15 000 rads), and immature DC were loaded with apoptotic LCL according to a method previously described (19); (III) PBMC were plated and stimulated as in protocol I. At day +12, selection of IFN-c + T cells by immunomagnetic microspheres coated with anti-IFN-c monoclonal antibody (mAb) (Miltenyi, Bergisch Gladbach, Germany), and restimulation with autologous LCL were performed according to protocol I; (IV) PBMC were plated at the same conditions as protocol I, in the presence of 10 ng/mL rhIL-7 and 10 pg/mL rhIL-12 (both, R&D Systems).…”

“…Recent studies have demonstrated that it is feasible to activate EBV-specific CTLs in adult EBV-naïve individuals, by prolonged LCL stimulation (18) or by priming with dendritic cells (DC) loaded with apoptotic/necrotic LCL (19). However, priming of EBV-specific CTL in children resulted in the expansion of CD4+ T cells (18).…”