Two aspects of this project were emphasized in the past year: studies elucidating effects on the veto response of enhancing signaling through the MHC target, and efforts to develop a B cell specific expression system. In the first project, we examined whether the veto phenomenon could be enhanced by improving signaling through the foreign MHC target of allospecific T cells. cDNA of MHC class I Dd, fused to that of the zeta (z) chain of the TCR, a potent signaling molecule, generated cell surface expressed Dd-z protein when transfected into Jurkat T cells. The zeta moieity of the fusion molecule conferred enhanced functional activity, as Jurkat cells expressing Dd-z produced high levels of IL-2, comparable to that elicited through TCR, on cross-linking of Dd by mAb, whereas production from Jurkats expressing Dd alone was paltry. Lymphocyte populations of transgenic mice (FVB: H-2q), in which expression of Dd-z was restricted to lymphocytes through genetic targeting (Dd-z mice), proliferated vigorously to antibody cross-linking and to engagement by allospecific parental lymphocytes in a one way MLR (FVB anti-Dd-z), in which neither FVB responders, nor Dd-z """"""""stimulators"""""""" were irradiated. Strikingly, such one way MLRs resulted in a dramatic reduction in Dd specific kill compared to MLRs in which the Dd-z stimulators were irradiated. To assess whether activated Dd-z cells could have reduced Dd specific kill by eliminating cells that bound to Dd (i.e. a veto mechanism), MLR activated Dd-z cells were incubated with EL-4 tumors, which bind avidly to Dd via Ly49A receptors. MLR activated Dd-zs killed EL-4s at very high levels, and the high level of kill was attributable solely to Dd-zs, as killing was maintained even when the FVB """"""""responders"""""""" were irradiated and therefore incapable of generating CTL. These studies indicate that activated Dd-z cells can efficiently eliminate cells binding to Dd and suggest that the severe reduction in Dd specific kill by FVBs stimulated with unirradiated Dd-z cells, may be due to elimination of FVB responders. The phenotype of the potent Dd-z killers was not of typical CTL, in that they were CD3-,TCR-,CD4-, and CD8-, but were positive for Thy1.1, Ly6c, CD5, and Dd-z, a phenotype that pertains to a unique population of cells present in high numbers in Dd-z mice. Such cells offer the distinct advantage of having potent veto ability without the capacity to induce GVHD. Based on extensive observations of the tolerogenicity of B cell APC, from our laboratory as well as others, the goal of the second project was to construct a B cell specific expression system. Because Ig heavy chain expression constructs mediate expression to a great degree in T, as well as B cells, we constructed a B cell expression system based on light chain elements, with the thought that the later rearrangement of light chain elements may allow for more selective B cell expression. Our expression system consists of two light-chain enhancers and a J-chain promoter. In preliminary studies, the construct appears to mediate high level expression in B tumor lines, whereas expression appears lower in a T cell line. To assess activity in different cell lineages, transgenic mice will be generated in which MHC class I Dd will be placed into the expression cassette. Furthermore, we are exploring the possibility of altering retroviral vectors such that they are more specifically and highly expressed in tolerogenic lymphocyte populations. Mcfarland, H.I.,Hansal, SA, Morris, D, Sechler,Weissman, A. Love, P.,and AS Rosenberg. Effects of enhanced signaling through MHC Manuscript in preparation.
