Retargeting of a T cell line by anti MAGE-3/HLA-A2 alpha beta TCR gene transfer.

BACKGROUND: The T cell receptor (TCR) is an heterodimeric protein on the cell membrane of cytotoxic T cells (CTLs). In CTLs TCRs mediate the recognition of target cells through interaction with specific, MHC class I presented peptides. MATERIALS AND METHODS: As a model system to show proof of principle we chose the Jurkat/MA cell line and the HLA-A2.1 binding MAGE-3 derived peptide 271-279, as target specificity. RESULTS: We show that this cell line can be successfully transduced with the dicistronic retroviral vector (LZRS) containing cDNAs encoding for the complete alpha and beta chains of the selected TCR. Following retroviral transduction, Jurkat/MA cells do express the anti-MAGE-3 TCR on their membrane. The transduced TCR is functional as travoductants are successfully triggered, upon stimulation with T2 cells or MAGE-3+ melanoma cells loaded with the MAGE-3 peptide. CONCLUSION: We conclude that TCR gene transfer is possible and it represents a powerful therapeutic tool for the genetical modification of T calls of patients sullering from cancer.

Renal-cell carcinoma-specific lysis by cytotoxic T-lymphocyte clones isolated from peripheral blood lymphocytes and tumor-infiltrating lymphocytes.

Melanoma and renal-cell carcinoma (RCC) are generally considered to be relatively immunogenic tumor types in humans. In the case of melanoma, many major histocompatibility complex (MHC) class I-restricted tumor-specific cytotoxic T lymphocytes (CTL) have been isolated from either tumor-infiltrating lymphocytes (TIL) or autologous peripheral blood lymphocytes (PBL). In contrast, such CTL have only incidentally been described in the case of RCC. It has often been reported that TIL lines isolated from RCC display non-MHC-restricted and non-specific activity. Here, we report the isolation and characterization of tumor-specific CTL from PBL of one RCC patient and from TIL of another RCC patient. CTL clones 263/17 and 263/45, isolated from the PBL of patient LE-9211, were restricted by HLA-B7. CTL clone 5E, isolated from the TIL of patient LE-8915, was restricted by HLA-B37. The autologous RCC cell lines were efficiently lysed by the CTL clones, whereas normal epithelial cells of the proximal tubuli matched for the restriction element and K562 were not. From a panel of allogeneic RCC cell lines, CTL 5E recognized MZ-1940-RCC. Reactivity to allogeneic RCC sharing HLA-B7 was also observed with CTL 263/17 and 263/45, both of which could lyse the HLA-B7-positive cell line MZ-1851-RCC. Our data provide evidence that common tumor antigens are recognized by CTL on RCC.

Relevance of ultraviolet-induced N-ras oncogene point mutations in development of primary human cutaneous melanoma.

Intermittent or recreational exposure to sunlight is thought to contribute to development of human cutaneous melanoma. We investigated the incidence of ras oncogene mutation in human cutaneous melanoma in connection to sun-exposed body sites in the patient, using a large series of DNA samples derived from paraffin-embedded material as well as from fresh tumor samples and cell lines. We first show that, of the ras family, predominantly N-ras is activated (15%), whereas rarely H-ras or K-ras are mutated. The occurrence of N-ras mutations correlates with continuous exposure to sunlight of the primary tumor site. Of all tumors initiated on chronically sun-exposed body sites, 26% contained mutated N-ras, in contrast to 0% of sun-protected melanomas. Melanoma lesions obtained from patients from North or Central Europe contained fewer N-ras mutations (12%) as compared with patients from Australia (24%). Mutations were specifically associated with nodular melanoma and to a lesser extent with lentigo malignant melanoma. N-ras mutations did not correlate with metastasis or survival parameters. This study identifies a subset of cutaneous melanomas that contain in the primary lesion ultraviolet-induced N-ras mutations, which are maintained through further progression.

c-myc-induced natural killer cell sensitivity of human melanoma cells is reversed by HLA-B27 transfection.

In human melanoma, activation of the c-myc oncogene results in locus-specific down-modulation of the HLA-B antigen expression. Moreover, overexpression of c-myc induces an increase of natural killer (NK) sensitivity of the tumor cells. To show that this effect on susceptibility to NK cells is mediated by the down-modulation of the HLA-B expression rather than by the activation of the oncogene, we supertransfected a c-myc transfectant with the gene encoding the HLA-B27 protein. The resulting supertransfectants with HLA-B27 surface expression were all less sensitive to NK cells than their parental cell line and showed a level of resistance equal to the original melanoma cell line with low c-myc expression. This indicates that the induction of NK sensitivity by c-myc activation in human melanoma cells is mediated through down-modulation of the HLA-B expression. These data also imply differential effects of HLA-A and HLA-B molecules on lysis by NK cells, because the level of NK susceptibility can apparently be defined by the level of HLA-B, irrespective of a substantial level of HLA-A expression present in the tumor cells.

Enhancement of the lytic activity of cloned human CD8 tumour-infiltrating lymphocytes by bispecific monoclonal antibodies.

We have isolated tumour-infiltrating lymphocytes (TIL) clones from a patient with renal cell cancer. The cloning frequency and the effector function were measured. No difference in cloning frequency (r2 = 0.97, frequency = 1:13) was observed between TIL expanded with allogeneic versus autologous feeder cells. Sixty-four clones expanded with autologous feeder cells and 37 clones expanded with allogeneic feeder cells were assayed for cytolytic activity on an autologous primary renal cell carcinoma (RCC) culture, an allogeneic RCC line, and on the K562 and Daudi cell lines. Most of these clones were also phenotyped. Although TIL clones expressing cytotoxic activity for RCC lines could be generated with both feeder cell preparations, none of the clones tested showed specificity for cells from autologous primary RCC cultures. However, in the presence of relevant bispecific MoAbs (alpha OC/TR) all CD8+ TIL clones tested could be induced to lyse autologous RCC cultures. Furthermore, the cytolytic activity of all CD8+ clones tested against allogeneic RCC lines could be induced or further enhanced by alpha OC/TR or CD3/G250 bispecific MoAbs. In contrast, none of the CD4+ clones tested showed lytic activity. Quantitatively the cytotoxic response in the presence of alpha OC/TR or CD3/G250 of CD8+ TIL clones against G250+ and MOv18+ cell lines appears to be associated with the level of antigen expression on the target cells. Our results suggest that: (i) expansion of TIL with allogeneic or autologous feeder cells does not effect the lytic profile of the clones; (ii) the use of bispecific MoAbs may overcome a lack of specificity of cytotoxic T lymphocytes.

[Transient complete AV block as a sequela of Borrelia myocarditis]. / Passagere komplette AV-Blockierung als Folge einer Borrelienmyokarditis.

A 49-year-old man suddenly developed dyspnoea, sweating, fever (up to 38.5 degrees C), vertigo and angina. After emergency admittance to hospital the ECG showed 3 degrees A-V block, requiring temporary pacemaker insertion. The patient reported that a month before he had been bitten, probably by a tick. Serological tests demonstrated a recent Borrelia infection (rise of IgG antibody titre to 1:2048, IgM antibody titre to 1:128). Coronary angiography excluded any haemodynamically significant coronary heart disease as a cause of the conduction disorder. Myocardial biopsy showed changes pointing to a past myocarditis. This suggested Borrelia infection as the cause of the complete A-V block. Under treatment with broad-spectrum antibiotics for 15 days the fever subsided and the ECG became normal. Shortly before discharge, an elevated pulmonary wedge pressure on 150 W exercise indicated persistence of mild left-ventricular failure.