The initiation and perpetuation of autoimmune disorders involves the abnormal presentation of self-antigen to rare auto-reactive T cells. Little is known about the factors that lead to inappropriate antigen presenting function by dendritic cells (DC). As part of immunoregulation, DC are normally subject to apoptosis once T cells are activated, therefore the development of apoptosis-resistant DC, which indeed have been detected in some autoimmune syndromes, may be an important contributing factor leading to autoimmunity. To examine this possibility we propose to engineer DC expressing anti-apoptotic genes and determine what impact this genetic manipulation has on initiating immune responses. We will use a well-established adoptive transfer animal model that permits the in vivo examination of interactions between engineered DC and normally rare naive, activated or tolerized antigen-specific T cells.
The specific aims of this proposal are as follows: 1. To evaluate the ability of DC over-expressing the ability anti-apoptotic genes to resist apoptotic stimuli in vitro. Bcl-XL, mC10, 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 systematically examined. Therefore, DC lines and primary DC will be stably and transiently transfected, respectively, 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. Engineered DC will be adoptively transferred into mice containing defined numbers CD4+ transgenic T cells specific for antigen-MHC II complexes exclusively presented on the DC transfectants. The fate of engineered DC will be tracked in draining lymph nodes over time to determine their life span. 3. To assess the ability of apoptosis-resistant DC to induce antigen-specific immune responses in vivo. The ability of antigen-presenting, apoptosis-resistant DC to induce a broad-spectrum of immune responses upon transfer to naive mice and their impact on T cell activation in vivo will be compared to those generated from mice receiving control DC transfectants DC life- span and its effect on T cell activation and fate may lead to new insights into the pathogenesis of autoimmunity. Thus, in future experiments, the ability of these engineer4ed DC to activate normally tolerized T cells will be directly examined in autoimmune animal models. Furthermore, the ability to genetically manipulate C biology in general, and longevity specifically, has important implications for developing strategies to engineer DC-based immunotherapies for ameliorating autoimmune disease.
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