Cytokine-Independent Proliferation of IL-2-Nonproducing CTL Clones in Association with High TCR-Signal Transduction Responses

Alloreactive CTL clone D2-23 proliferated in response to antigenic cells without IL-2 production. Among subclones of D2-23, the F1 but not F2 clone proliferated in response to soluble aCD3 or PMA, although both clones proliferated in response to immobilized aCD3, antigenic cells or soluble aCD3 plus costimulatory cells. The difference in responsiveness between F1 and F2 was not caused by distinct expression of CD3 or Fc receptors. Cyclosporin A, which totally blocks IL-2 production of Th1 cells, barely or only partially inhibited PMA- or aCD3-induced proliferation of F1. F1 did not produce cytokines for proliferation of F2 in response to soluble aCD3. Tyrosine phosphorylation developed for various proteins of F1 and F2 at the levels apparently correlated to the extent of cell proliferation when the cells were stimulated with soluble aCD3 or PMA. The proliferative responsiveness of F1 and F2 to the described stimulators was maintained by stimulation with IL-2 plus antigenic cells, or even IL-2 alone, but was decreased during resting culture or by stimulation with immobilized aCD3. These results show evidence of a new TCR-linked mechanism for CTL proliferation that is independent of costimulatory cell- or cytokine-mediated signaling, but is originally prepared by prior stimulation with IL-2.     

TCR-Independent Induction of Low Responsiveness by Chemically Fixed Cells in Alloreactive CTL Clones and Its Prevention through Cognate Cell-Cell Interaction

We established BALB/c-derived CD8⁺ CTL clones D2-22 (V_β 6⁺), D2-23 (V_β 8⁺) and D2-24 (V_β 8⁺) specific for B10.D2 minor H antigen. D2-22 and D2-23 proliferated without producing IL-2 in response to X-ray-irradiated antigenic cells, Con A, aCD3, PMA and IL-2. Paraformaldehyde-fixed antigenic spleen cells neither induced proliferation in the presence of costimulatory cells nor inhibited responses to irradiated antigenic cells added simultaneously. Unlike the previously reported results with IL-2-producing CTL clones and Th1 clones, the fixed antigenic cells failed to induce antigen-specific unresponsiveness in these IL-2-nonproducing CTL clones. Instead, the responsiveness of these clones to fresh stimulation was found to be reduced severely after 2 days in the culture added with either antigenic or syngeneic fixed cells. Induction of their antigen-nonspecific low responsiveness by the fixed cells was prevented by adding irradiated syngeneic cells into the culture or even by increasing the concentration of responder D2-23 cells. Close contact of D2-23 and irradiated syngeneic cells was required to prevent the reduction of the responsiveness, although this cognate cell-cell interaction could be replaced by exogenously added IL-2 or PMA. Cytolytic and tumor cell growth inhibitory activities of D2-23 were also reduced by incubation with the fixed cells, which was prevented by the addition of irradiated syngeneic cells. These findings showed the unique properties of IL-2-nonproducing CTL clones in signal requirements for maintaining normal responsiveness for proliferation and cytolytic activity.     

Temporal variation of the static electric field inside an animal cage

The temporal variation of a static electric field inside an animal cage was investigated with a newly developed small, simple field meter. The field inside the cage was found to be highly dependent on the surface conductivity of the dielectric material. As the surface of the cage became dirty because of animal occupancy, the static electric field inside it became considerably smaller from the moment the field was turned on. Clean cages also modified the static electric field inside them, the field decaying from an initial to a much lower value over several hours. The mechanism of field attenuation for both cases is surface leakage. Surface leakage for a clean cage takes place much more slowly than for a dirty cage. This was confirmed by measuring DC insulation resistance. To examine this phenomenon further, the field in a metal cage with high electrical conductivity was measured. The static electric field inside the metal cage was also found to be reduced. An improved cage design that avoids these problems, is suggested for the study of the biologic effects of static electric fields.     

Effects of iron and desferrioxamine on Rhizopus infection

To investigate the association among iron, desferrioxamine, and a Rhizopus infection, the influence of iron and/or desferrioxamine on experimental mucormycosis in mice was examined. All mice pretreated with iron, desferrioxamine, or a combination of iron and desferrioxamine died within 5 days after the inoculation of R. oryzae. In the mice fungal lesions were observed in the brain which resembled human cerebral mucormycosis. By contrast, the mortality in the control mice with R. oryzae was 20% through the 3-week experimental period. Therefore, it was demonstrated that iron as well as desferrioxamine administration markedly promotes the growth of R. oryzae. The increased susceptibility to R. oryzae was considered to be due to increased serum iron in the animals pretreated with iron only; however, pretreatment with desferrioxamine did not affect the amount of serum ion. Thus, the data suggest that desferrioxamine acts as a siderophore to R. oryzae and exerts an adverse effect on mucormycosis. This study has shown that the presence of iron and desferrioxamine enhances the virulence and pathogenicity of R. oryzae by serving as a growth factor.