Microsatellite, restriction fragment-length polymorphism, and sequence-specific oligonucleotide typing of the tumor necrosis factor region. Comparisons of the 4AOHW cell panel.

The location of the TNF and other genes in the central MHC and their possible relevance to disease susceptibility provided an impetus to develop useful typing markers. The 4AOHW undertook to assess the various markers available, including DNA sequence-based systems. A panel of well-characterized lymphoblastoid cell lines were typed by Nco I RLFP analysis, SSO typing, and TNF microsatellite typing. RFLP and SSO typing were relatively reproducible as judged by the blind replicates. The two techniques provided the same results with only one exception, and it would be reasonable to prefer SSO typing because of its advantages in terms of cost and time. Microsatellite typing was much more discriminating but, as expected, less robust in that some discrepancies were apparent. As a result of the workshop and subsequent testing, alleles and haplotypes were allocated to most cells within the 4AOHW panel, including 10W cells typed in previous studies. While there was evidence that microsatellites may be relatively stable, they have the potential to identify recent mutations within ancestral haplotypes.

The induction of immune responses to murine malignant mesothelioma by IL-2 gene transfer.

Stable IL-2 transfectant clones have been derived from two non-immunogenic murine malignant mesothelioma (MM) cell lines to investigate the induction of protective antitumor immunity to MM. AC29-IL-2 transfectant clones grew at a slower rate in vivo than the parental cell line or a transfectant control clone but all inoculated mice developed tumours despite the continued ability of the tumour cells to express IL-2. Tumour development after inoculation of AB1-IL-2 transfectants varied, the degree of in vivo inhibition (40-100%) being directly related to the rate of IL-2 secretion of the transfectants. When mice which had rejected the AB1-IL-2 transfectants were challenged with parental AB1 cells, a proportion (16-70%) of mice from each group remained tumour free at least 45 days after challenge (naive mice developed tumours within 26 days). The inhibition of growth of the initial inoculum of AB1-IL-2 transfectants was independent of CD4+ and CD8+ cells, consistent with the demonstration of non-specific cytotoxic activity by splenocytes from mice inoculated with the IL-2 transfectants. These data suggest that IL-2 expression by MM cells is capable of generating in vivo immunity to the tumour. This immunity may be relatively weak or may be subject to down-regulation so that consistent rejection of unmodified tumour cells is not achieved. Genetic modification with combinations of genes, including IL-2 and B7-1, will be necessary for reliable generation of protective immunity to MM.

Transfection of the gene for B7-1 but not B7-2 can induce immunity to murine malignant mesothelioma.

Transfection of the genes encoding the co-stimulatory molecules B7-1 and B7-2 has enhanced the development of immunity to a variety of experimental tumors, although most of these were inherently immunogenic. We have determined the effect of expression of these genes on the induction of immunity to 2 non-immunogenic murine malignant mesothelioma (MM) cell lines (AC29 and AB1). We had previously shown that B7-1 transfection into AC29 delayed but did not prevent tumor development by certain of the transfectant clones. Here we demonstrate that over-expression of B7-1 can inhibit tumor development by certain AB1-B7-1 clones, that inhibition of transfectant growth is dependent on CD4+ and CD8+ T cells and that mice that reject some of these transfectant clones are capable of rejecting subsequent inocula of the parental cell line, AB1. The transfectant clones can generate tumor-specific cytotoxic T cells. By contrast, expression of B7-2 in several clones derived from either AB1 or AC29 had no significant effect on the development of tumors in vivo. Our data are consistent with data from other systems that show differences in the effect of modification by B7-1 or B7-2 on the modulation of anti-tumor immune responses. They demonstrate that such modifications can induce protective immunity against an MM cell line but confirm the intra- and inter-tumoral heterogeneity in the effect of genetic modification on the induction of immunity. Our observations are relevant to human MM because these cell lines have been derived from asbestos-induced tumors and share many properties with human cell lines of the same histological type.