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We have studied the effects of human rIL-12 on the proliferation and generation of cytotoxic activity in human CTL precursors. Purified human blood CD8+ T lymphocytes were stimulated overnight with immobilized alpha-CD3 and cultured 3 to 4 additional days under various conditions. The addition of IL-12 resulted in a marked (10- to 20-fold), dose-dependent, augmentation of cytotoxicity per cell with a smaller (2-fold) increase in cell number. IL-12 augmentation of proliferation and cytotoxicity of CD8+ T cells was not inhibited by a mAb to the p55 subunit of the IL-2 receptor (alpha-Tac) at a concentration sufficient to block the activity of exogenously added IL-2, indicating that the activity of IL-12 did not require IL-2. Addition of IL-12 at the time of alpha-CD3 activation or 1 day later was highly effective at augmenting cytotoxicity, whereas delayed addition of IL-12 (day 2 or 3) resulted in a smaller increase in CTL activity with no increase in cell number. IL-12 at all doses tested synergized with low dose IL-2 in inducing the proliferation and differentiation of CD8+ T cells. The synergistic effect was not blocked by adding neutralizing serum to IFN-gamma. In contrast to this synergistic effect, IL-12 significantly inhibited the proliferation observed in the presence of higher concentrations of IL-2 (4,500 and 13,500 pg/ml). An inhibitory effect of IL-12 was also observed when IL-12 was added to CD8+ T lymphocytes 3 days subsequent to activation with alpha-CD3 and IL-2. This broad set of potent effects of IL-12 on CD8+ T cell responses suggests that IL-12 may play an important immunoregulatory role on CTL development in vivo and may be a useful tool for manipulating this process in vivo for investigational and immunotherapeutic purposes.

The effects of IL-7 on the generation of human CTL in alloantigen-, virus-, and lectin-stimulated systems were examined. Addition of IL-7 at the onset of cultures resulted in marked (up to 80-fold) augmentation of cytotoxicity accompanied by smaller (1.5- to 4-fold) increases in total lymphocyte number. Studies of CTL development in purified lectin-stimulated CD8+ T cell populations demonstrated that IL-7 could act directly on the CD8+ lymphocyte subset to augment cytotoxicity. In MLC, the IL-7-induced enhancement of cytotoxicity was found to be mediated primarily by the CD8+ subpopulation of lymphocytes. Late addition of IL-7 (day 5 of 7) resulted in an increase in cytolytic activity that was associated with little or no increase in total or activated CD8+ lymphocyte number indicating that IL-7 may act as a differentiation factor for human CTL. A role for endogenous IL-7 in CTL development was suggested by the observation that addition of neutralizing antiserum to IL-7 to MLC at initiation (or 5 days thereafter) resulted in decreased levels of cytotoxicity. These results indicate that IL-7 can exert major up-regulatory effects on human CTL development and suggest that these effects are both proliferative and differentiative.

The effect of endogenous and exogenous IL-4 on the generation of influenza virus-specific cell-mediated immunity was examined. When added at the onset of the culture, IL-4 augmented both cluster of differentiation (CD)8+ lymphoproliferation and MHC-restricted, influenza virus-specific cytotoxicity. When added 5 or 6 days after initiation of cultures, IL-4 was highly effective at augmenting cytotoxicity, whereas no augmentation of proliferation was observed. This disassociation of the effect of IL-4 on lymphoproliferation and cytotoxicity indicated that IL-4 was providing a late signal in CTL generation. Studied at the level of CTL precursor maturation in microcultures, IL-4 was found not to increase cytotoxicity but to be required, in some cases, for the generation of cytotoxicity. Endogenous IL-4 production was observed and demonstrated to be important because neutralizing antiserum to IL-4 suppressed CTL development. In contrast to the effects of IL-4 when added later to the cultures, pulsing the lymphocytes with IL-4 before, or shortly after, exposure to antigen resulted in suppression of the CTL response. These results indicate that IL-4 has differentiative, proliferative, and suppressive effects on cell-mediated immune responses.