: Genetically engineered subsets of the dendritic cell (DC) family, such as Langerhans cells (LC), hold tremendous promise as immuno-therapeutic agents in the development of DC-based vaccines for cancer and infectious diseases. However, engineered DC quickly undergo cell death by immune-mediated apoptosis in vivo, crippling the fill potential of DC-based therapies. We propose that expressing anti-apoptotic genes within DC will inhibit apoptosis and extend their life span in vivo. Thus we will test if extending the in vivo life span of engineered DC will amplify the engineered antigen-specific immune response. The effect of these genes on the life span of an LC-derived DC cell line and their ability to modulate immune responses will be investigated utilizing the well-established 3A9 transgenic animal model that permits in vivo examination of interactions between DC and normally rare naive or activated antigen-specific T cells.
The specific aims of this proposal are as follows: 1. To evaluate the ability of anti-apoptotic genes to confer resistance to apoptotic stimuli within DC in vitro. Bcl-XL, CrmA and baculovirus p35 block steps in biochemically separate pathways of apoptosis. Some have been implicated in altering DC longevity, but their effects on DC have not been examined. Therefore, DC will be transfected with anti-apoptotic genes and their ability to resist apoptotic stimuli in vitro will be tested. 2. To evaluate the effect of anti-apoptotic genes on altering the life span of transfected DC in vivo. In vivo studies will utilize adoptive transfer of engineered DCs into mice containing defined numbers CD4+ T cells (obtained from 3A9 transgenic mice) that recognize specific antigen-MHC II complexes exclusively presented on the DC transfectants. The effect of anti-apoptotic genes on extending DC life span in vivo will be determined. 3. To assess the impact of apoptosis-resistant DC on immune responses in vivo. The transfected anti-apoptotic DC abilities to induce a broad-spectrum of immune responses or to develop immune disorders upon transfer to naive mice and their impact on I cell activation in vivo will be examined. 4. To quantify the relationship of DC longevity to the magnitude of the DC-engineered immune response. An inducible """"""""suicide"""""""" gene (iCasp3) will be super-transfected into anti-apoptotic DC as well as wild-type DC. At different times after adoptive transfer into mice DC will undergo apoptosis in vivo by exposing mice to the inducing agent. Thus, the minimal DC life span required to generate a defined immune response will be determined. Moreover, these experiments will lay the foundation toward achieving exogenous control of engineered DC gene expression and, in this case, longevity in vivo.
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