T cells are critical components of cell-mediated immunity. They can aid in the production of high-affinity antibodies by B cells or directly kill virally infected cells. A greater understanding of the biochemical and cellular events which regulate T cell activation has come from the study of downstream genes which are targets of T cell activation, including lymphokines such as IL-2. Recent reports from this laboratory and others now point to a role for the much-studied Akt kinase, a target of PI-3 kinase, in NF-kappaB activation and co-stimulation of IL-2 transcription. As CD28-mediated co-stimulation of IL-2 is known to occur through effects on NF-kappaB, this led to the hypothesis that Akt overexpression may be able to replace the CD28 co-stimulatory signal under some conditions. Overexpression of wild type Akt could replace the CD28 signal for induction of transcription in a cell line. This also holds true for primary T cells. Thus, infection of CD28 deficient T cells with a retrovirus encoding activated Akt is able to restore IL-2 (and also IFN-gamma, but not IL-4 or IL-5) production. These results are consistent with a requirement for PI-3 kinase and Akt activation in the production of IL-2 by normal T cells. The goals of this proposal are two-fold: first, to extend the observation that Akt is able to selectively restore production of IFN-gamma and IL-2 (but not Th2 cytokines) by CD28 deficient cells; second, to study the effects of inducible Akt activation in vivo, eventually applying the findings to a murine autoimmune disease model.
Aim 1 : Determine which regions of CD28 are responsible for upregulation of Thl and Th2 cytokines. Others have described mutations in the CD28 cytoplasmic tail that can abolish IL-2 upregulation. Such mutants will be investigated, with or without Akt co-expression, for their ability to upregulate a spectrum of cytokines in primary T cells, using retroviral reconstitution. These studies will be performed in collaboration with Drs. John Imboden and Abul Abbas.
Aim 2 : Examine the effects of inducible Akt expression in vivo. I am currently generating founder mice that should carry an inducible allele of Akt, under the control of a tetracycline response element. These mice will be used to examine the effects of Akt in primary, naive T cells that have developed normally. Tetracycline can then be used to induce Akt expression, after which T cell proliferation and cytokine production to model antigens can be followed. In addition, mice treated chronically with tetracycline will be followed for signs of autoimmune disease, to see if signaling through Akt is sufficient to break peripheral tolerance.
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