Phenotypic characterization and analysis of allogeneic T cell responses in ZAP-70 dominant negative transgenic mice.

Antigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70-SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4+ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.

In vivo augmentation of IFN-gamma with a rIL-12 human/mouse chimera: pleiotropic effects against infectious agents in mice and rats.

A heterodimer containing the mouse 35 kDa and human 40 kDa subunit of IL-12 was expressed in COS cells (cIL-12). Administration of 25-200 U of the cIL-12-COS supernatant to mice twice daily for 2 days augmented spleen cell IFN-gamma production in response to IL-2 and peritoneal macrophage activity (superoxide and nitrites) as compared to animals receiving mock transfected supernatants. cIL-12 also increased levels of IFN-gamma in serum but most dramatically following an LPS injection (50-fold over controls). Animals pretreated with cIL-12 suffered enhanced mortality following challenge with the Gram negative organism E. coli but enhanced survival or clearance following infection with the Gram positive organisms L. monocytogenes and S. aureus. Although daily treatment of mice with cIL-12 following an intranasal influenza A infection elevated levels of IFN-gamma in the bronchioalveolar lavage fluid three-fold over controls, neither prophylactic or therapeutic treatment with the same dose level decreased viral titres in the lung. In addition, no effect was observed in animals infected with encephalomyocarditis virus or respiratory syncytial virus. Therefore, cIL-12 is a potent in vivo augmentor of IFN-gamma production. It has differential effects on infectious disease depending on the invading organism and time of administration; being efficacious for intracellular bacteria but ineffective at the same dose levels against viral diseases.