[Computer-controlled laser irradiation unit for studies of light-induced processes in cell cultures]. / Computergesteuerte Laserbestrahlungseinheit für Untersuchungen lichtinduzierter Prozesse an Zellkulturen.

For the photodynamic treatment of tumours, synergistic effects of photosensitizing substances and light (today usually laser light) are used. With the aim of optimizing photosensitizing drugs and therapy, the effects of light and drug dose were studied in cell experiments. To automate and standardize such in vitro experiments, a laser irradiation chamber was developed. Cells cultured from tumour cell lines are placed on micro-titre plates or in petri dishes, together with the photosensitizer, and subsequently irradiated in the irradiation chamber with a well-defined dose of laser light of a wavelength corresponding to the absorbance of the photosensitizing agent. The plates or dishes are irradiated from below. In this way, light dose errors due to refraction from the meniscus of the cell suspension as occurs with irradiation from above, are avoided. During irradiation, speckle effects on the underside of the plates or petri dishes lead to variation in irradiation. A vibrator keeps the light transmission fibre and thus speckle pattern in motion, guaranteeing a homogeneous irradiation of the cells.

Induction of a novel cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri.

Although herpesvirus saimiri-transformed T lymphocytes retain multiple normal T-cell functions, only a few changes have been described. By subtractive hybridization, we have isolated a novel cellular gene, ak155, a sequence homolog of the interleukin-10 gene. Specifically herpesvirus saimiri-transformed T cells overexpress ak155 and secrete the protein into the supernatant. In other T-cell lines and in native peripheral blood cells, but not in B cells, ak155 is transcribed at low levels. AK155 forms homodimers similarly to interleukin-10. As a lymphokine, AK155 may contribute to the transformed phenotype of human T cells after infection by herpesvirus saimiri.

The superantigen-homologous viral immediate-early gene ie14/vsag in herpesvirus saimiri-transformed human T cells.

Herpesvirus saimiri C488 transforms human T lymphocytes to stable growth in culture. The growth-transformed human T cells harbor the viral genome in a nonintegrated episomal form without production of virus particles. In these cells, virus gene expression was previously found to be confined to the transforming genes stpC and tip. In order to analyze virus gene expression in more detail, we applied a subtractive hybridization technique and compared stimulated virus-transformed cells with uninfected parental T cells of the same donor. A number of known T-cell activation genes were isolated. Viral stpC/tip cDNAs were enriched after subtraction. In addition, the viral immediate-early, superantigen-homologous gene ie14/vsag was represented by numerous cDNA clones that comprised the entire spliced transcript. Whereas a weak basal expression of ie14/vsag was detected by reverse transcription-PCR only, the phorbol ester-induced transcripts were readily shown by Northern blotting. ie14/vsag, which before had been classified as a major immediate-early gene of herpesvirus saimiri, is localized within a highly conserved region with extensive homologies to the cellular genome. Mutant viruses without the ie14/vsag gene are replication competent and fully capable of transforming human and marmoset T cells. Since ie14/vsag is transiently expressed after stimulation, it may increase T-cell proliferation in an activation-dependent and superantigen-like but apparently Vbeta-independent way.

Regulation of the herpesvirus saimiri oncogene stpC, similar to that of T-cell activation genes, in growth-transformed human T lymphocytes.

Herpesvirus saimiri strain C488, a T-cell tumor virus of New World primates, transforms human T lymphocytes to stable interleukin-2-dependent growth without need for further stimulation by antigen or mitogen. The transformed cell lines show the phenotype of activated mature T cells and retain many essential features of the primary parental cells, e.g., antigen specificity. In contrast to transformed New World monkey T cells, the human lines do not support lytic growth of the virus, even after chemical stimulation. Here we show that many viral genes remain silent during episomal persistence. However, the viral oncogene stpC is predominantly transcribed and translated to a stable cytoplasmic protein of 20 kDa that is heterogeneously expressed in individual cells. This 1.7-kb mRNA is bicistronic, encoding also Tip, a viral protein interacting with the T-cell-specific tyrosine kinase Lck. stpC/tip transcripts are heavily induced upon stimulation by mitogen or phorbol ester. Block of protein synthesis does not abolish transcription: treatment with cycloheximide greatly induces stpC/tip mRNA levels. Thus, this gene complex is regulated similarly to early T-cell activation genes. Constitutive and induced expression engage different transcription start sites. The T-cell regulation of the viral genes stpC and tip may contribute to the T-cell tropism of growth transformation by herpesvirus saimiri.

Functional phenotype of transformed human alphabeta and gammadelta T cells determined by different subgroup C strains of herpesvirus Saimiri.

Based on sequence divergence in the transformation-relevant region, herpesvirus saimiri strains are classified into three subgroups. Only members of subgroup C transform human T lymphocytes to continuous interleukin-2-dependent growth in culture. In this study, human cord blood T cells were immortalized by using different subgroup C strains (C488, C484, and C139). The resulting T-cell lines represented different types of T-cell clones. They were either CD4+ or CD8+ and expressed either the alphabeta or the gammadelta type of T-cell receptors. If transformed by the same virus strain, alphabeta and gammadelta clones were similar with respect to viral persistence, virus gene expression, proliferation, and Th1-type cytokine production. However, major differences were observed in T cells immortalized by different subgroup C strains. Strain C139 persisted at low copy number, compared to the high copy number of prototype C488. The transformation-associated genes stpC and tip of strain C488 were strongly induced after T-cell stimulation. The homologous genes of strain C139 were only weakly expressed and not induced after activation. After CD2 ligation, the C488-transformed T cells produced interleukin-2, whereas the C139-transformed cells did not. Correspondingly, the C139-transformed T cells were less sensitive to cyclosporin A. Sequence comparison from different subgroup C strains revealed a variability of the stpC/tip promoter region and of the Lck-binding viral protein Tip. Thus, closely related subgroup C strains of herpesvirus saimiri cause major differences in the functional phenotype of growth-transformed human T cells.

Herpesvirus saimiri-transformed macaque T cells are tolerated and do not cause lymphoma after autologous reinfusion.

Human T cells are transformed in vitro to stable growth after infection with herpesvirus saimiri subgroup C strain C488, and they retain their antigen-specific reactivity and other important functional features of mature activated T lymphocytes. The virus persists as nonintegrating episomes in human T cells under restricted viral gene expression and without production of virus particles. This study analyzes the behavior of herpesvirus-transformed autologous T cells after reinfusion into the donor under close-to-human experimental conditions. T cells of 5 macaque monkeys were transformed to stable interleukin-2 dependent growth and were intravenously infused into the respective donor. The animals remained healthy, without occurrence of lymphoma or leukemia for an observation period of more than 1 year. Over several months virus genomes were detectable in peripheral blood cells and in cultured T cells by polymerase chain reaction. In naive control animals, a high-dose intravenous infection rapidly induced pleomorphic peripheral T-cell lymphoma. In contrast, monkeys were protected from lymphoma after challenge infection if they had previously received autologous T-cell transfusions. High levels of antibodies against virus antigens were detectable after challenge infection only. Taken together, herpesvirus-transformed T cells are well tolerated after autologous reinfusion. This may allow us to develop a novel concept for adoptive T-cell mediated immunotherapy.

T-cell lymphoma caused by herpesvirus saimiri C488 independently of ie14/vsag, a viral gene with superantigen homology.

The immediate-early gene ie14/vsag of herpesvirus saimiri has homology with murine superantigens. We compared the pathogenesis of infection with either ie14/vsag deletion mutants or wild-type virus C488 in cottontop tamarin monkeys (Saguinus oedipus). Two weeks after infection, all animals developed acute T-cell lymphomas independently of the presence of the viral ie14/vsag gene.

The interleukin-17 gene of herpesvirus saimiri.

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.