Studies on the mechanism of suppression of primary cytotoxic responses by cloned cytotoxic T lymphocytes.

We have shown in the accompanying companion paper that cloned cytotoxic T lymphocytes (CTL) can serve as veto cells in vitro, suppressing primary cytotoxic activity directed against antigens expressed by those cloned CTL but not against third party antigens. We now explore the mechanism of this antigen-specific suppression by cloned CTL, using as a model system the ability of G4, a BALB.B anti-H-2Dd CTL clone, to specifically suppress a primary in vitro anti-H-2b CTL response. G4 cells do not constitutively secrete a suppressor factor, because suppression cannot be mediated by supernatants removed from G4 cells at a time when they are routinely used as veto cells. Furthermore, medium removed from cultures suppressed by G4 will not suppress, indicating that the veto cell function of G4 is not mediated by soluble factors. Full suppression of primary anti-H-2b CTL responses requires that G4 be present throughout the 5-day mixed lymphocyte culture (MLC). Removal of G4 during the first 3 days of MLC results in a drastic reduction in the level of antigen-specific suppression, with a slight but reproducible loss of suppression after veto cell removal on day 4. The addition of G4 during the course of an ongoing MLC reveals that maximal suppression requires the presence of veto cells during the first 24 to 48 hr of culture. Thus, G4 cells must be present both early and late in an MLC to exert maximal veto cell suppression. Several experiments suggest that G4-induced veto cell activity is unlikely to be due to cytolysis of CTL precursors which are capable of recognizing G4. G4 cannot specifically recognize these CTL precursors, and G4 cells are inefficient at lectin-mediated lysis of non-tumor cell targets. Furthermore, we show that G4 cells cannot lyse CTL which recognize them. Finally, dilutions of anti-clonotypic antibodies which completely block both lectin-mediated and specific cytolysis by G4 do not block (and in fact enhance) G4-mediated veto cell activity.