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.