Yukiko Nakata

Radiosensitivity of CD45RO+ memory and CD45RO- naive T cells in culture.

Radiosensitivities of various human T-cell subsets were investigated by a proliferation assay and by a single-cell gel electrophoresis assay. Each T-cell subset was purified using a cell sorter and was induced to proliferate by ionomycin and interleukin 2. Unsorted T cells showed biphasic dose-survival curves, indicating the heterogeneity of T cells in terms of radiosensitivity. Purified CD4+ helper and CD8+ killer T cells showed similar biphasic dose-survival curves. Hence both T-cell subsets were composed of cells of different radiosensitivity. The T-cell subsets belonging to different activation stages such as CD45RO+ memory and CD45RO- naive T cells had different dose-survival curves. The former was more radiosensitive than the latter. The high radiosensitivity of CD45RO+ cells was also demonstrated by single-cell gel electrophoresis after irradiation. This is the first demonstration that a particular cell surface marker on T cells is correlated with greater radiosensitivity.

In vivo induction of cytotoxic T lymphocytes specific for a single epitope introduced into an unrelated molecule

Cytotoxic T lymphocytes (CTLs) recognise antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules on virus-infected cells. The formation and transportation of antigenic peptides to class I MHC in the cells are multi-step reactions known as antigen processing. In order to design a good DNA vaccine, it is important to dissect the specificity of antigen processing. Here we describe the construction of an epitope-based plasmid vector as a device to investigate antigen processing in transfected cells. The epitope-based plasmid vector was constructed by insertion of an epitope-encoding minigene into the lacZ gene. We used a CTL epitope on influenza A virus nucleoprotein (NP366-374 epitope) as a model. Upon transfection, the epitope-based plasmid vector induced the expression of NP epitope antigenically as well as immunogenically. Immunization of mice with plasmid-transfected cells was able to induce NP epitope-specific CTLs in vivo. Moreover, the plasmid vector functioned as a gene vaccine; NP epitope-specific CTLs were primed in vivo upon transfection of the vector into dermis by electroporation. The results suggest that this epitope-based DNA delivery system may provide a new strategy for in vivo induction of epitope-specific CTLs to investigate antigen processing and presentation.

Characteristic association between K-ras gene mutation with loss of heterozygosity in X-ray-induced thymic lymphomas of the B6C3F1 mouse.

Purpose : To elucidate the characteristics of radiation carcinogenesis, the spectra of K- and N- ras oncogene mutations, loss of heterozygosity (LOH) and their association in X-ray-induced thymic lymphomas (TL) were determined by comparing with those of N -ethyl- N -nitrosourea (ENU)-induced and spontaneously occurring TL. Materials and methods : TL that arose in untreated, X-ray-irradiated and ENU-treated B6C3F1 mice were examined both for K- and N- ras mutations by PCR-SSCP and DNA sequencing and for LOH by PCR with polymorphic microsatellite markers. Results : (1) ras gene mutations were found in a proportion of TL from X-ray-exposed (~20%) and ENU-treated (30-40%) mice while no ras gene mutations were found in spontaneous TL. N- ras mutations were rare. (2) The spectrum of ras gene mutations was diverse and seemed to differ little between X-ray-induced and ENU-induced TL, even though there was a higher frequency of ras mutations in ENU-induced TL that clustered to K- ras codon 12. (3) The X-ray-induced TL showing K- ras mutation were associated with LOH on chromosome 6, while those showing no K- ras mutation were associated with high frequency of LOH on chromosomes 4, 11 and 12. Conclusion : These results demonstrate that, in the B6C3F1 mouse TL, X-ray-induced lymphomagenesis showed both the co-expression, yet low occurrence of allelic imbalance on chromosome 6 and K- ras mutation, and exclusive expression of frequent allelic imbalance on chromosomes 4, 11 and 12 and K- ras mutation.