This project is in the final phases of completion. We have focused on a cell population generated in transgenic mice in which signaling through class I MHC Dd was enhanced by fusing the TCR zeta (z) chain to the extracellular domains of Dd. Transgenic mice expressing high copy numbers of the Dd-z fusion molecule, which was targeted to lymphocytes by a CD2 promoter, have markedly diminished numbers of CD4+ and CD8+ T cells and a reciprocally high percentage of CD3-CD5+ cells which are TCR-CD4-CD8- cells but which are of T cell lineage as they express Thy 1.1,CD44, and Ly6C. Interestingly they express supraphysiologic level of Dd. These cells appear similar to memory CD8+ CTL, not only by cell surface appearance, but also functionally, as they have abundant cytolytic activity which can be activated by 1) TCR hybridomas recognizing cognate peptide in the context of Dd 2) mAb to Dd, and 3)cellular recognition of Dd by spleen cells from FVB mice primed to Dd. These cells potently kill cells that bind to Dd such as EL-4 cells which bind Dd through cell surface Ly49A. The mechanism by which CTL activity is mediated is being investigated, but does not appear to depend on Fas ligand expression or TRAIL expression. Furthermore, these memory CTL like cells also potently block the devlopment of an anti-Dd CTL reponse by both naive and primed FVB lymphocytes. Thus, these CD3-CD5+ T lineage cells represent an ideal type of a """"""""veto"""""""" cell in being able to diminish even potent memory Dd specific CTL responses, yet because they lack TCR, they also lack the ability to induce GVHD. Regarding the second aspect of this project, the construction of an expression system that would induce high level expression on B cells in vivo, despite generating a construct that induced high level expression on B cell tumor lines in vitro, consisting of two light-chain enhancers and a J-chain promoter, we were unsuccessful in producing transgenic mice expressing such a construct. Additional studies involving direct transfection of primary lymphocyte populations ex-vivo are being pursued. Mcfarland, H.I.,Hansal, SA, Morris, D, McVicar, D,Weissman, A. Love, P.,and AS Rosenberg. Effects of enhanced signaling through MHC Manuscript in preparation.
