Mechanisms of selective immunosuppressive effects of indoleamine 2, 3-dioxygenase and borrelidin

Indoleamine 2, 3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor that has been considered as a promising candidate in down-regulating alloimmune responses. IDO expression generates a tryptophan-deficient environment that selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) underlying these selective effects of IDO is not elucidated. In this doctoral research project, we hypothesize that different sensitivity of immune cells versus skin cells to IDO-induced tryptophan-deficient environment is due to the differential activity of general control non-derepressible-2 (GCN2) kinase stress-responsive pathway and its inhibitor, protein IMPACT homolog, in these cells. Considering the selective effect of tryptophan-deficiency on immune cells, we also proposed that borrelidin, a small molecule agent that mimics the effects of amino acid deprivation, may provide potential therapy for acute lymphoblastic leukemia (ALL) via its selective apoptotic effects. To this end, we investigated whether borrelidin selectively inhibits the proliferation of malignant ALL cell lines and studied the mechanism by which this drug acts. Three specific objectives were accomplished in this study. We first showed that in the presence of IDO, higher activation of GCN2 in immune cells leads to apoptosis and is due to the very low or undetectable expression of IMPACT in these cells. Our gain- and loss-of-function findings suggest that high expression of IMPACT in non-immune cells acts as a protective mechanism against IDO-induced apoptosis. In the next phase, in order to stably express IDO in grafts to prolong their survival, a pure population of IDO-expressing bystander fibroblasts was generated using two simple yet effective lentiviral-based approaches. Finally, borrelidin was used as a novel strategy in treating ALL. We showed that borrelidin treatment potently and selectively inhibits proliferation, induces apoptosis and mediates G₁ arrest in ALL cell lines and that borrelidin treatment in these cells is correlated with activation of the GCN2 pathway. The findings presented in this thesis collectively demonstrate the mechanism of IDO action and pave the way towards application of IDO as an immunosuppressive factor in development of long-lasting non-rejectable allografts. Our findings also reveal the potential application of borrelidin in treating ALL.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.Medicine, Faculty ofSurgery, Department ofNon UBCReviewedFacult