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Spleen cells obtained from BALB/c mice 4 days after allosensitization to C57BL/6 spleen cells via footpad injection suppressed the in vitro generation of BALB/c cytotoxic lymphocytes (CL) against C57BL/6 spleen cells in mixed leukocyte cultures (MLC). Suppressor activity was demonstrated by spleen cells at 4 and 7 days but not at 2, 10, or 14 days after allosensitization and was abolished by treatment with anti-Thy-1,2 serum and complement. A weak and transient cytotoxic response directed against the sensitizing alloantigen was associated with suppressor spleen cell populations, but was dissociated from suppressor function by two experimental approaches. First, increasing stimulatory cell concentration in MLC did not competitively diminish the suppressor activity; rather, the magnitude of suppression increased as the stimulatory cell concentration was increased. Second. BALB/c suppressor cells generated in vivo by either H-2b or H-2k alloantigens suppressed CL responses generated simultaneously against both alloantigens in vitro. CL responses generated against one or the other H-2 haplotype in vitro were suppressed only by suppressor cells activated by that haplotype. Therefore, splenic suppressor cells activated by alloantigen in vivo required antigen-specific restimulation in vitro; thereafter, responder cells syngeneic with the suppressor cell were rendered hyporesponsive to alloantigens by an antigen-nonspecific mechanism.

Generation of cytotoxic T lymphocytes (CTL) in mixed leukocyte cultures was suppressed by a factor elaborated by alloantigen-activated T cells. This suppressor factor, CTL-TsF, in contrast to a factor that suppresses proliferative responses in mixed leukocyte reactions (MLR-TsF), was effective only when added during the first 24 hr of a 6-day-culture period. Moreover, removal of CTL-TsF 24 hr after culture initiation failed to restore CTL responses. CTL activity could be rescued from suppressed cultures, however, by addition of 2-mercaptoethanol on days 3 or 4. Similarly, transfer of nonadherent cells at 3 or 4 days from cultures treated with CTL-TsF to cultures of adherent cells initiated in control factor restored CTL responses. Mixing experiments with cells pulsed with CTL-TsF for 4 hr at culture initiation identified a target of CTL-TsF as a Thy-1 negative cell that was adherent to plastic and to Sephadex G-10. Suppression was not due to interference with physiologic accessory cell function, but more likely was accomplished via a negative signal from CTL-TsF-pulsed cells. The results thus suggest that CTL-TsF acts early, but reversibly, in the CTL differentiative process via a second suppressor effector cell, possibly a macrophage.

Suppression of in vitro cytotoxic lymphocyte (CL) responses was mediated by soluble factor(s) produced when in vivo alloantigen-activated suppressor cells were re-exposed to alloantigen in vitro. Elaboration of suppressor factor (SF) was T cell dependent and was optimal 7 days after alloantigen injection. Suppressor factor failed to inhibit CL generation when alloantigen-primed cells rather than normal spleen cells were used as responders. Moreover, SF added at day 3 of incubation rather than at culture initiation was also ineffective, suggesting that suppression probably occurs during antigen induction or early differentiation. Additionally, suppression was abrogated by the presence of 2-mercaptoethanol. Studies combining SF and CL responder cells from a variety of H-2 disparate mouse sdrains revealed that suppression of CL responses: 1) was not alloantigen specific; 2) did not require H-2 homology between responder and suppressor strains; and 3) could not be demonstrated with CBA/J mice. Although CBA/J CL responses were not suppressed by any SF preparation, allo-sensitized CBA/J spleen cells did elaborate SF that inhibited BALB/c CL responses.