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.

The influence of interleukin (IL)-12 and IL-4 on the differentiation of naive CD4+ T cells was studied in an accessory cell-free in vitro system. Dense CD4+ T cells were purified from unimmunized mice and activated using immobilized anti-CD3 monoclonal antibodies (mAb) in the presence of IL-4, IL-12, or a combination of both cytokines, and restimulated after 6 days by re-exposure to anti-CD3-coated culture wells. T cells initially activated in the presence of IL-4 produced substantial amounts of IL-4 and trace amounts of interferon (IFN)-gamma after restimulation at day 6 with plate-bound anti-CD3 mAb. By contrast, T cells primed in the presence of IL-12 produced high levels of IFN-gamma and only minimal amounts of IL-4, thus indicating that IL-12 and IL-4 by acting directly on stimulated naive CD4+ T cells support the development of TH1 and TH2 cells, respectively. When naive CD4+ T cells were stimulated in the presence of IL-12 together with IL-4 in comparable concentrations, the effect of IL-12 on TH1 differentiation was largely inhibited by IL-4. On the other hand, IL-12 exerted no inhibitory effect on IL-4-induced TH2 differentiation but rather enhanced the production of IL-4 after restimulation of the respective T cells. Decreasing amounts of IL-4 in combination with a high level of IL-12 led to an increasing production of IFN-gamma by the emerging T cells and, simultaneously, to a relatively high production of IL-4. These data were confirmed by time-course experiments which revealed that the delayed addition of IL-4 to IL-12-primed T cell cultures resulted in a gradual restoration of IFN-gamma production whereas in parallel the secretion of IL-4 was not reduced over a wide period of delay (6-72 h). These results, therefore, demonstrate that (a) IL-4 dominates the effect of IL-12, (b) IL-12 promotes the development of TH1 cells; however, in the presence of IL-12 and relatively high levels of IL-4 also the development of TH2-like cells is slightly but significantly enhanced by IL-12, and (c) high amounts of IL-12 in combination with relatively low levels of IL-4 give rise to a T cell population that upon rechallenge exhibited a cytokine profile resembling that of TH0 cells.

Activation of naive dense CD4+ T cells by plate-bound anti-CD3 antibodies favors the development of Th1 cells which, upon re-stimulation, produce significant amounts of IFN-gamma but no IL-4. However, co-activation of such naive T cells in the presence of IgE [anti-dinitrophenyl (DNP)]-loaded bone marrow-derived mast cells (BMMC) on plates coated with anti-CD3 antibodies and DNP-BSA led to the development of IL-4-producing Th2 cells. The same result could be observed if irradiated (800 rad) BMMC were applied as co-stimulators. Moreover, BMMC could be replaced by the supernatant of IgE-activated BMMC suggesting that a soluble mediator, presumably IL-4, was responsible for this effect. This assumption was substantiated using neutralizing anti-IL-4 antibodies which abolished the BMMC-mediated Th2 development in all cases. Addition of IL-12, a cytokine that was shown to antagonize the Th2-promoting effect of IL-4 in vivo, could not inhibit the development of IL-4-producing T cells, but gave rise to a T cell population which produced relatively high amounts of IL-4 and IFN-gamma. Since BMMC represent the in vitro equivalent of mucosal mast cells these data suggest that IgE-activated mucosal mast cells can bias an emerging T cell dependent immune response towards a Th2 dominated reaction by the initial production of IL-4.

CD8+ T cells recognize antigenic peptides presented in the context of MHC class I. They play a key role in cellular immunity and are crucial for longterm protective immunity to many infectious diseases. The quest for new and enhanced vaccines requires improved means for identification of relevant antigens and the epitopes present within these. While there are several algorithms available for epitope prediction (all of which work to differing degrees of success), the definition of actual MHC class I-binding epitopes is very reliant on time-consuming and difficult to perform functional assays using often very limited biological material. The iTOPIA assay is quick and easy to perform and determines real-binding to MHC class I molecules. It provides an excellent platform for screening and elimination of potential epitopes and identification of novel epitopes prior to validation with a relevant functional assay.

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