The influence of the cytokine interleukin-12 (IL-12) on humoral immune responses was studied in vivo. CBA/J mice immunized with protein antigens (keyhole limpet hemocyanin, phospholipase A2) adsorbed to aluminum hydroxide (Alum) develop a Th2-like immune response characterized by the production of large amounts of IgG1 as well as some IgE but little IgG2a, IgG2b and IgG3 antibodies. IL-12 is a cytokine that promotes the development and the activation of Th1 cells. Th1 cells are involved in the induction of cellular immunity, which is characterized by low or absent antibody production. On the other hand, some Th1-like immune responses are associated with a strong antibody production of the IgG2a, IgG2b and IgG3 subclasses. Thus, we investigated whether treatment with IL-12 would down-regulate the humoral immune response or stimulate antibody production of the IgG2a, IgG2b and IgG3 subclasses. We observed that: 1) administration of IL-12 to mice together with protein antigens adsorbed to Alum strongly enhanced the humoral immune response by increasing the synthesis of antigen-specific antibodies of the IgG2a, IgG2b and IgG3 subclasses 10- to 1000-fold. The synthesis of IgG1 was not or only slightly (2-5-fold) enhanced, whereas that of the IgE isotype was suppressed. 2) These effects of IL-12 were observed when high (10 micrograms, 100 micrograms) or low doses (0.1 microgram) of antigen were used for immunization. 3) Titration of IL-12 in vitro revealed that IgG2a is strongly up-regulated over a wide dose range of IL-12 (10 to 1000 ng/day). 4) The effects of IL-12 in vivo are at least partially interferon (IFN)-gamma-dependent because an anti-IFN-gamma mAb in combination with IL-12 prevented most of the enhanced IgG2a production. 5) Mice receiving IL-12 showed a strong up-regulation of IFN-gamma but no inhibition of IL-5 synthesis by spleen cells activated ex vivo with antigen. These results suggest that IL-12 is a potent adjuvant for enhancing humoral immunity to protein antigens adsorbed to Alum, primarily by inducing the synthesis of the complement-fixing IgG subclasses 2a, 2b and 3.

The recently discovered cytokine interleukin (IL)-12 is a heterodimeric protein of two disulfide-bonded subunits of 35 and 40 kDa. IL-12 has multiple effects on T cells and natural killer (NK) cells. In particular it appears to be a major factor for the development of cellular immunity. So far activity of the single subunits alone has not been described, however their expression is regulated independently. In this report we demonstrate for the first time that the mouse IL-12 subunit p40 (IL-12p40) specifically antagonizes the effects of the IL-12 heterodimer in different assay systems. The proliferation of mouse splenocytes activated by phorbol ester and IL-12 was inhibited by IL-12p40, whereas the proliferation induced by phorbol ester and IL-2 was not affected. Furthermore, the synthesis of interferon (IFN)-gamma by mouse splenocytes activated with IL-2 and IL-12 was suppressed by IL-12p40. Purified mouse splenic CD4+ T cells produced IFN-gamma upon activation with plate-bound anti-CD3 monoclonal antibody which was enhanced more than tenfold in the presence of IL-12. In this system IL-12p40 inhibited only the enhancement caused by IL-12 but not IFN-gamma synthesis of CD4+ T cells stimulated with anti-CD3 alone. Moreover, IL-12p40 inhibited the effects of IL-12 on differentiated T helper type 1 (Th1) cells. IFN-gamma production by Th1 cells induced in a T cell receptor-independent way by macrophages and IL-2 or macrophages and IL-12 was greatly reduced by IL-12p40 providing evidence for the endogenous synthesis of IL-12 in the Th1 cell, macrophage and IL-2 co-cultures. The specificity of inhibition was clearly demonstrated in the homotypic aggregation assay of Th1 cells. Incubation of Th1 cells with either IL-2 and IL-12 or IL-2 and tumor necrosis factor induces LFA-1/ICAM-1-dependent aggregation. Only IL-2 + IL-12 but not IL-2 + tumor necrosis factor-induced aggregation was inhibited in a dose-dependent manner by IL-12p40. Thus, the IL-12 subunit p40 appears to be a specific inhibitor for the IL-12 heterodimer.

At least two subsets of CD4+ T helper cell lymphocytes termed Th1 and Th2 exist in the mouse and probably in humans. They are characterized by the secretion of different lymphokines and by their functional behavior. Dysregulated expansion of one or the other subset may be one reason for the development of certain diseases. Thus, it is of importance to define the signals involved in the differentiation and activation of the two Th cell subsets. It is known and has been confirmed in this report that the cytokine interleukin (IL)-1 acts on Th2 cells but not on Th1 cells. We now report that a previously identified cytokine which was provisionally termed T cell stimulating factor is identical with IL-12 and exhibits a reciprocal behaviour to IL-1. IL-12 has several effects on Th1 cells. It can induce the proliferation of certain Th1 cells in combination with IL-2. Synthesis of interferon (IFN)-gamma by Th1 cells can be triggered by IL-2 plus IL-12. In contrast to the IFN-gamma production observed after T cell receptor (TcR) CD3 stimulation of Th1 cells with lectin Concanavalin A the IFN-gamma production induced by IL-12 + IL-2 is insensitive to the immunosuppressive drug cyclosporin A. Furthermore, IL-12 enhances the TcR/CD3-induced synthesis of IFN-gamma of several Th1 clones. Finally, IL-12 (+IL-2) induces homotypic cell aggregation of Th1 clones. This type of cell aggregation depends on the participation of LFA-1 and ICAM-1 molecules. In all activation systems with Th1 cells no effect of IL-1 was demonstrable. In contrast, only IL-1 but not IL-12 served as a co-stimulatory signal for several Th2 cell lines activated via the TcR/CD3 complex.

It was observed in vitro and in vivo that both interferon (IFN)-gamma and interleukin (IL)-12 can promote the development of T helper type 1 (TH1) cells. Since IL-12 was shown to be a costimulator for the production of IFN-gamma by T or natural killer (NK) cells, IL-12 might play only an indirect role in TH1 differentiation by providing IFN-gamma which represents the essential differentiation factor. Using anti-CD3 monoclonal antibody (mAb) for activation of naive CD4+ T cells in the absence of accessory cells we could demonstrate that costimulation by IFN-gamma alone results only in marginal TH1 development. Similarly, IL-12 in the absence of IFN-gamma is only a poor costimulator for inducing differentiation towards the TH1 phenotype. Our data indicate that both cytokines are required to allow optimal TH1 development and that IL-12 has a dual role, it promotes differentiation by direct costimulation of the T cells and also enhances the production of IFN-gamma which serves as a second costimulator by an autocrine mechanism. Another cytokine that was reported to favor TH1 differentiation in certain experimental systems is transforming growth factor (TGF)-beta. With naive CD4+ T cells employed in this study TGF-beta strongly inhibited the production of IFN-gamma triggered by IL-12 as well as the IL-12-induced TH1 development. When TGF-beta was combined with anti-IFN-gamma mAb for neutralization of endogenous IFN-gamma the TH1-inducing capacity of IL-12 was completely suppressed.

We have previously shown that certain bone marrow-derived mast cell (BMMC) lines proliferate in response to a mast cell growth-enhancing activity (MEA) that is distinct from interleukin (IL) 3 and IL 4. Here we provide evidence that MEA is identical with the recently cloned mouse T cell growth factor P40. The evidence is as follows: (a) recombinant P40 displayed all the biological activities ascribed to MEA: it supported the growth of MEA-sensitive BMMC lines, it induced IL 6 secretion by these cells, and it enhanced survival of primary mast cell cultures; (b) highly purified MEA stimulated the growth of P40-dependent cell lines; (c) a rabbit monospecific antiserum directed against P40 specifically inhibited the action of MEA on BMMC; (d) specific binding sites for P40 were detected on BMMC and (e) MEA competed with P40 for binding to P40-dependent T cells, indicating that the two molecules interact with the same receptor. These observations further extend the range of biological activities ascribed to P40 and warrant its proposed designation as IL9.