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.