Date of Award

10-19-2009

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

George Tellides

Second Advisor

John P Geibel

Abstract

TRYPTOPHAN DEPLETION BY INDOLAMINE 2,3-DIOXYGENASE (IDO) CONTRIBUTES TO MEDIAL IMMUNOPRIVILEGE IN GRAFT ARTERIOSCLEROSIS. Amanda M. Silverio, Madison C. Cuffy, and George Tellides. Interdepartmental Program in Vascular Biology and Transplantation and the Departments of Surgery and Immunobiology, Yale University, School of Medicine, New Haven, CT. Introduction: Since blood vessels and their endothelial lining are major stimulators and targets of the graft rejection response, the study of the differential immune properties of vascular cells is needed for the proper application of human vessels in the transplant setting. Experimental models of atherosclerosis and graft arteriosclerosis show leukocytes selectively infiltrating the intima and adventitia while sparing the media. We explored the role of vascular smooth muscle cells (VSMCs) in mediating the observed immunosuppression and attributed it to the expression of indoleamine 2,3-dioxygenase (IDO) by VSMCs. IDO, in response to interferon-gamma (IFN-γ) signaling, catalyzes the first and rate-limiting step in the oxidative catabolism of tryptophan, an essential amino acid, and generates kynurenine metabolites. We further investigated the role of IDO in vascular immune regulation using both in vivo and in vitro studies. Methods: Segments of human coronary arteries from explanted hearts of cadaveric organ donors or cardiac transplant recipients were interposed into the infrarenal aortae of SCID/beige mice and harvested after five weeks for histologic analysis. RT-PCR reactions were prepared with pre-developed assay reagents for IDO, tryptophanyl-tRNA synthetase (WRS), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression level of each target was normalized to that of GAPDH. Co-culture experiments were performed by placing 2x105 endothelial cells (ECs) or VSMCs along with CFSE-labeled CD4+ T-cells in 24-well plates and treating with IFN-γ at 100 ng/mL for 3 days. The cultures were maintained in 5% CO2 at 37°C for up to 9 days. T-cell proliferation was assessed by fluorescence-activated cell sorting (FACS) analysis of CFSE dilution after counterstaining with PE-labeled mouse anti-human CD4 antibody. Interleukin-2 (IL-2) levels were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Results: Vessel grafts showed that medial sparing by allogeneic T-cells is associated with IDO expression by VSMCs. Exposure to the T-cell-derived cytokine, IFN-γ, led to 100-fold greater expression of IDO mRNA in graft VSMCs than in ECs or CD4+ T-cells (2.00, 0.02, and undetectable IDO/GAPDH mRNA ratios, respectively). An immunoregulatory effect was shown when IFN-γ-treated VSMCs decreased CD4+ T-cell proliferation and IL-2 production (levels decreased by 98% and 90%, respectively), and addition of the IDO inhibitor, 1-methyl-tryptophan, led to a reversal of the inhibition. Inhibition was not seen with ECs or untreated VSMCs. CD4+ T-cell anergy was replicated by tryptophan depletion, but not by the addition of tryptophan metabolites: kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. Tryptophan supplementation rescued T-cell proliferation in a dose-dependent fashion. Conclusions: We confirmed that vascular cells, namely VSMCs, could regulate adaptive immune responses by preferentially expressing IDO to inhibit CD4+ T-cell activation and clonal expansion. We also characterized IDO regulation of T-cells in the medial wall by showing that the depletion of tryptophan in the microenvironment inhibited CD4+ T-cell proliferation. Considering that the immune system relies on tryptophan regulation as a method to selectively modulate T-cell infiltration in graft vessel walls, the expression of IDO and the availability of tryptophan serve as potential therapeutic targets in the management of graft arteriosclerosis.

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