Interferon-gamma differentially regulates susceptibility of lung cancer cells to telomerase-specific cytotoxic T lymphocytes.

There is accumulating evidence that peptides derived from the catalytic subunit of human telomerase reverse transcriptase (hTERT) are specifically recognized by CD8+ cytotoxic T lymphocytes. We investigated the cytotoxicity of a human leukocyte antigen (HLA)-A*2402-restricted hTERT-derived peptide 461-469 (hTERT461)-specific CD8+ T-cell clone, designated as K3-1, established from a healthy donor by repetitive peptide stimulation. This clone exhibited cytotoxicity against 4 out of 6 HLA-A24-positive lung cancer cell lines with positive telomerase activity but not 4 HLA-A24-negative examples. When the target cells were pretreated with 100 U/ml of interferon (IFN)-gamma for 48 hr, the susceptibility to K3-1 increased with PC9 cells but unexpectedly decreased with LU99 cells. However, in both cell lines, the expression of molecules associated with epitope presentation such as HLA-A24, transporters associated with antigen processing, low molecular weight polypeptide 7 and proteasome activator 28 was similarly increased after IFN-gamma treatment. Results of CTL assays using acid-extracted peptides indicated that the epitope increased on PC9 cells but not on LU99 cells after IFN-gamma treatment. Semi-quantitative reverse transcriptase polymerase chain reaction disclosed that the expression of hTERT was attenuated in LU99 but not in PC9 cells, accounting for the decreased cytotoxicity mediated by K3-1. The attenuation of the hTERT expression and K3-1-mediated cell lysis after IFN-gamma treatment was also observed in primary adenocarcinoma cells obtained from pulmonary fluid of a lung cancer patient. Our data underline the utility of peptide hTERT461 in immunotherapy for lung cancer, as with other malignancies reported earlier, and suggest that modulation of hTERT expression by IFN-gamma needs to be taken into account in therapeutic approach.

Characterization of Epstein-Barr virus (EBV)-positive NK cells isolated from hydroa vacciniforme-like eruptions.

Recently, the involvement of Epstein-Barr virus (EBV) in hydroa vacciniforme (HV)-like eruptions has been suggested. To elucidate the role of EBV in this disease, we isolated EBV-infected cell clones from peripheral blood mononuclear cells (PBMC) and the skin lesions of a patient with HV-like eruptions; cells isolated from PBMC were designated SNK-12, and those from the eruption SNK-11. Both cells expressed CD16, CD56, and HLA-DR and had germline configurations of the T-cell receptor and the immunoglobulin genes, indicating that the cell clones were of NK cell lineage. The analysis of EBV terminal repeats indicated that the cells were monoclonal, had identical clonality, and originated from EBV-positive cells in the PBMC and eruption. Both clones expressed EBNA-1, but not EBNA-2. Although LMP-1 was weakly detected in SNK-11, no LMP-1 was detected in SNK-12. Interestingly, EBV-infected cells required less IL-2 for in vitro growth in the later phase of this disease and this appeared to correlate with the expression of LMP-1, suggesting that the proliferative capacity of the EBV-positive NK cells increased during the time course of the disease, and LMP-1 expression might be responsible for that. This is the first report of the isolation of EBV-infected cells from the skin lesions of HV-like eruptions and strongly suggests that the HV-like eruption in the patient was caused by clonal NK cells with latent EBV infection.

Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas, chronic active EBV infection and hydroa vacciniforme-like eruptions.

In this study, we describe the cytological and cytogenetic features of six Epstein-Barr virus (EBV)-infected natural killer (NK) cell clones. Three cell clones, SNK-1, -3 and -6, were derived from patients with nasal T/NK-cell lymphomas; two cell clones, SNK-5 and -10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK-11, was from a patient with hydroa vacciniforme (HV)-like eruptions. An analysis of the number of EBV-terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK-cell lymphomas but also those isolated from CAEBV and HV-like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T-cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV-positive NK cells, which proliferated in the diseased individuals. The results showed that EBV-positive NK cells in malignant and non-malignant lymphoproliferative diseases shared common cytological and cytogenetic features.

Preferential expansion of Vgamma9-JgammaP/Vdelta2-Jdelta3 gammadelta T cells in nasal T-cell lymphoma and chronic active Epstein-Barr virus infection.

We recently established an Epstein-Barr virus (EBV)-positive gammadelta T-cell line from a nasal T/natural killer (NK)-cell lymphoma (Nagata H, Konno A, Kimura N, Zhang Y, Kimura M, Demachi A, Sekine T, Yamamoto K, Shimizu N: Characterization of novel natural killer (NK)-cell and gammadelta T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus. Blood 2001, 97:708-713). Subsequently, we established two novel EBV-positive gammadelta T-cell lines from the peripheral blood of patients with chronic active EBV infection. Analysis of the terminal repeat of EBV showed that the three cell lines consisted of monoclonal populations, and flow cytometry showed that they had a common phenotype of gammadelta T cells: CD3(+) CD4(-) CD8(-) CD16(-) CD19(-) CD56(+) CD57(-) HLA-DR(+) T-cell receptor (TCR) alphabeta(-) TCR gammadelta(+). Analysis for the expression of TCR by flow cytometry showed that all three cell lines were Vgamma9(+)/Vdelta2(+), but negative for VgammaI, Vdelta1, or Vdelta3 TCR. Southern blot analysis for TCR genes showed that the three cell lines had a common rearrangement of Vgamma9-JgammaP and Jdelta3 genes. Polymerase chain reaction and sequence analysis of the junction between Vdelta and Jdelta genes revealed that the Jdelta3 genes were rearranged with the Vdelta2 genes. In contrast, none of the EBV-negative gammadelta T-cell lines, Molt-14, Peer, or Loucy, which were analyzed for controls, had Vgamma9 or Vdelta2 TCR, or a rearrangement of Jdelta3 genes. These results indicated that Vgamma9-JgammaP/Vdelta2-Jdelta3(+) gammadelta T cells were preferentially affected by EBV and expanded in patients with nasal gammadelta T-cell lymphoma and chronic active EBV infection. Jdelta3(+) gammadelta T cells are known to be a very minor population in gammadelta T cells of peripheral blood, whereas Vgamma9-JgammaP/Vdelta2-Jdelta1(+) cells are the major population. The close association of EBV with this particular gammadelta T-cell population may provide a key to the etiology of EBV-positive lymphoproliferative diseases.