Shuhji Seki

Details of an isolation method for hepatic lymphocytes in mice

The liver comprises a unique lymphocyte population, i.e., extrathymic alpha beta T cells with TcR of intermediate intensity. In the present study, we attempted to determine what pretreatments were appropriate to isolate hepatic mononuclear cells (MNC) containing such intermediate alpha beta TcR cells in mice. Hepatic MNC were isolated from untreated mice and mice subjected to either bleeding or liver perfusion, and the intermediate alpha beta TcR cells in each preparation were identified. For reasons of simplicity, cell purity and cell yields, hepatic lymphocytes should be obtained from mice subjected to total bleeding. Additional information on extrathymic alpha beta T cells obtained by using the recommended method is also presented.

Activation of extrathymic T cells in the liver and reciprocal inactivation of intrathymic T cells by bacterial stimulation

We recently demonstrated that the liver might be a major site of extrathymic T cell differentiation, including both alpha beta and gamma delta T cells. This extrathymic pathway in the liver, which has a relatively minor role in normal young mice, is activated in mice under bacterial stimulation. In the present study, we investigated how the extrathymic and intrathymic T cell differentiations were mutually related in mice injected intravenously with 10(8) heat-killed Escherichia coli. Three days after stimulation, extrathymic T cells in the liver were observed to be prominently activated in terms of increases in the total number of cells yielded, spontaneous cell proliferation in in vitro culture, and intermediate alpha beta TCR cells. Intermediate alpha beta TCR cells were extrathymic T cells uniquely seen in the liver. However, at the same time intrathymic T cells were profoundly inactivated, showing decreases in the number of thymocytes (more than 90% atrophy), spontaneous cell proliferation, and dull TCR cells with double positive CD4+8+ phenotype. With time, these responses were reversed and normal states were regained. These results suggested that extrathymic and intrathymic T cells are always activated or inactivated in the opposite direction, and that the liver and the thymus are dynamic immune organs. It raises the possibility that the extrathymic T cell differentiation in the liver and the intrathymic T cell differentiation may be reciprocally regulated by certain factors.

Age-Associated Increase of CD5+ B Cells in the Liver of Autoimmune (NZB×NZW) F1 Mice

The liver has been demonstrated to be a major site for extrathymic differentiation of T cells. In this study, an identification of CD5+ B cells, which are responsible for the onset of autoimmune disease by virtue of autoantibody production, was performed in autoimmune (NZB × NZW) F1 mice. An age‐associated increase of CD5+ B cells was demonstrated in the liver of these mice. Although CD5+ B cells (i.e., CD5+IgM+ and CD5+B220+) constituted a minor population of hepatic mononuclear cells (MNC) (<5%) when mice were young (8 weeks), a large population of CD5+ B cells (10 to 30% of whole MNC) was identified in the liver of mice aged 25 to 30 weeks after the onset of disease. Such age‐dependent increase of CD5+ B cells was not observed in any other strains including NZB, NZW, C3H/He and BALB/c mice. The phenotype of hepatic CD5+ B cells was the same as that of CD5+ B cells in the peritoneal cavity and spleen, showing dull‐CD5, bright‐IgM and dull‐B220. High levels of CD5+ B cells were observed in the peritoneal cavity and liver, but not in the spleen nor in any other lymphoid organs in mice aged 30 weeks. Radioimmunoassay of autoantibodies in the 5‐day culture supernatants demonstrated that hepatic MNC were unable to produce any amounts of IgM‐ and IgG‐autoantibodies against double‐stranded DNA and single‐stranded DNA, despite the increased proportion of CD5+ B cells. On the other hand, peritoneal exudate cells produced only IgM‐, but not IgG‐, autoantibodies, whereas splenic cells were able to produce both IgM‐ and IgG‐autoantibodies. These results suggest that the liver might support the generation of the most primitive CD5+ B cells in these mice and that such generation increases as a function of age, probably resulting in the onset of autoimmune disease.

An appropriate in vitro culture condition for the induction of human TCRγδ+ cells by heat-killed bacteria

Abstract TCRγδ+ cells proliferated when MNC were stimulated with various heat-killed bacteria. We investigated here the culture conditions for their maximum proliferation. MNC were cultured for 6 days with Streptococcus pyogenes, and for 3 days with T cell mitogens, PHA and anti-CD3 mAb, in medium supplemented with various concentrations (0.05–50%) of human sera. TCRγδ+ and TCRγδ−CD2+3− double negative cells induced by Str. pyogenes required high concentrations of sera (>6%) for their proliferation. Moreover, increased sera (up to 50%) greatly augmented their proliferation. On the other hand, TCRαβ+ cell proliferation induced by T cell mitogens was supported by small concentration (even 0.1%) of the sera, and the addition of high concentrations of sera (>6%) somewhat suppressed responses. Similar serum requirement patterns were evident for the induction of cytotoxic cells. These results clearly demonstrated the existence of an appropriate culture condition for the proliferation of TCRγδ+ cells induced in vitro.