Clustered CD3/TCR complexes do not transduce activation signals after bispecific monoclonal antibody-triggered lysis by cytotoxic T lymphocytes via CD3

Bispecfic mAb (bsmAb) directed against the CD3/TCR complex and a tumor-associated Ag (TAA) induces CTL-mediated lysis of TAA+ target cells. We have investigated whether bsmAb-pretargeted CTL can enter multiple lytic cycles. BsmAb-pretargeted CTL retained bsmAb-targeted lytic capacity for at least 24 h when exposed to medium without TAA+ target cells. Exposure of bsmAb-pretargeted CTL to TAA+ target cells resulted in a rapid loss of bsmAb-targeted cytotoxicity of TAA+ or Fc gamma R+ target cells, although the CTL retained surface bsmAb. Moreover, addition of rabbit anti-mouse lg to these CTL did not induce calcium mobilization. These CTL still showed Ag-specific cytotoxicity and cytolysis of anti-CD3 mAb-expressing hybridoma cells. Readdition of bsmAb to CTL that had lost bsmAb-targeted cytotoxicity instantly restored the bsmAb-targeted lytic activity of the CTL. Hence, as in Ag-specific cytolysis, bsmAb-pretargeted CTL can enter multiple bsmAb-targeted cytolytic cycles. Surprisingly, exposure of bsmAb-pretargeted CTL to Fc gamma R+ cells did not result in loss of bsmAb-targeted cytolysis of TAA+ cells. Fluorescence microscopic analysis revealed that bsmAb-mediated interaction with TAA+ cells, but not with Fc gamma R+ cells, resulted in clustering of bsmAb-pretargeted CD3/TCR complexes on the CTL surface. On the basis of the observed correlation between clustered bsmAb-pretargeted CD3/TCR complexes and loss of bsmAb-targeted cytotoxicity, we hypothesize that clustered CD3/TCR complexes can no longer transduce signals.