Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies

Abstract

The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in EBV-positive Hodgkin disease and is a potential target for cytotoxic T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is low, and so far the generation of LMP1-specific CTLs has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DCs) for CTL stimulation, and we reasoned that an inactive, nontoxic LMP1 mutant (⌬LMP1) could be expressed in DCs and would enable the activation and expansion of polyclonal LMP1-specific CTLs. Recombinant adenoviral vectors expressing LMP1 or ⌬LMP1 were tested for their ability to transduce DCs. LMP1 expression was toxic within 48 hours whereas high levels of ⌬LMP1 expression were achieved with minimal toxicity. ⌬LMP1-expressing DCs were able to reactivate and expand LMP1-specific CTLs from 3 healthy EBV-seropositive donors. LMP1-specific T cells were detected by interferon-␥ (IFN-␥) enzyme-linked immunospot assay (ELISPOT) assays using the HLA-A2-restricted LMP1 peptide, YLQQN-WWTL (YLQ). YLQ-specific T cells were undetectable (less than 0.001%) in donor peripheral blood mononuclear cells (PBMCs); however, after stimulation the frequency increased to 0.5% to 3.8%. Lysis of autologous target cells by CTLs was dependent on the level of LMP1 expression. In contrast, the frequency of YLQ-specific CTLs in EBV-specific CTLs reactivated and expanded using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. IntroductionImmunotherapy with cytotoxic T cells (CTLs) is increasingly used to treat malignancies and viral infections. 1,2 For example, polyclonal Epstein-Barr virus (EBV)-specific CTLs have been used for the prevention and treatment of posttransplantation EBV-associated lymphoma (PTLD). 3 EBV-specific CTLs persisted long-term, reconstituted immunity against EBV, and produced antiviral and antilymphoma effects. We have also used EBV-specific CTLs to treat 13 patients with EBV-positive Hodgkin disease and, while the results have been promising, no patient with bulky disease has been cured. 1,4 One explanation for this failure is that current methods of EBV-specific CTL generation produce CTL lines that are dominated by clones reactive to EBV proteins not expressed in the malignant Reed-Sternberg cells (H-RS cells) of EBV-positive Hodgkin disease. 5 Only a limited number of EBV-derived antigens (EBNA1, BARF0, LMP1, and LMP2) are present in H-RS cells, 6,7 and the immunodominant response of CTL lines is against EBNAs 3A, B, and C, which are not expressed by the tumor cells. Of the EBV proteins expressed in H-RS cells, only LMP2 and LMP1 are potential targets for CD8 ϩ T cells, because EBNA 1 is mainly presented on major histocompatibility complex (MHC) class II molecules 8 and the level of BARF0 expression might be too low for CTL recognition. 9 Our group and others have recently reported the generation of LMP2-specific CTLs 10-12 ; however, for future clinical protocols it is desirable to generate CTLs against more than one tumor-as...

Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma

“…12,15 Briefly, Zs Green-expressing 143B cells were culture on sarcosphereforming media with different methotrexate concentrations for 4 h. Viable OS cells were sorted for ZsGreen and 1 Â 10 6 target cells were labeled with 0.1 mCi (3.7 MBq) 51 Cr and mixed with decreasing number of effector cells to give E:T ratios of 40:1, 20:1 and 10:1. Target cells incubated in complete medium alone of in 1% Triton X-100 were used to determine spontaneous and maximum 51 Cr release, respectively.…”

Epstein‐Barr virus (EBV) latent membrane protein‐1‐specific cytotoxic T lymphocytes targeting EBV‐carrying natural killer cell malignancies

“…To construct DLMP1 [20], PCR was performed using a sense primer 5 0 -aagcttgccaccATGAGTGACTGGACTGGA-3 0 , an antisense primer 5 0 -ttgaattcttagtcatagtagcttagctga-3 0 , and EBV strain B95-8 (NCBI accession No. V01555) cDNA as a template.…”

“…Intracellular staining of LMP1 Ag was performed as previously described with slight modification [20]. Briefly, electroporated cells were collected and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature.…”

“…They also showed the utility of a replication-incompetent adenovirus and a recombinant vaccinia virus encoding multiple LMP1 epitopes, successfully immunizing HLA-A2-transgenic mice and achieving inhibition of the growth of LMP1-transduced cells [18,19]. Gottschalk et al [20] reported effective induction of polyclonal LMP1-specific CTL using DC infected with a recombinant adenovirus expressing an N-terminally truncated, nontoxic LMP1 mutant. EBVpositive NK/T cell malignancies express EBNA1 and LMP1 as potential CTL targets [6,7], but it has not been demonstrated that such NK/T cells can process LMP1 and generate HLA-restricted epitopes, so that they are susceptible to CTL-mediated cytolysis.…”

“…We infer that mRNA-transduced APC might be suitable for induction of LMP1-specific CTL. In the present study, we applied a 43-amino acid Nterminal deletion mutant (DLMP1) as an Ag to reduce LMP1 cytotoxicity [26] and its potential to induce IL-10 production [20].…”