Potential Tolerizing Capacity of Human Dendritic Cells Featuring High Levels of Expression and Activity of the Tryptophan Metabolizing Enzyme Indoleamine 2,3 Dioxygenase.

Abstract

An enhanced tryptophan metabolism mediated by the enzymatic activity of indoleamine 2,3 dioxygenase (IDO) has recently been demonstrated to profoundly affect T cell responses. By the present study we explored whether human dendritic cells (DCs) displaying high IDO expression and activity, down-regulate allogeneic T cell responses. A comparison of lipopolysaccaride (LPS), interferon-γ (IFN-γ), and CD40L as DC maturation agents showed that most abundant IDO expression and activity in DCs was observed when immature DCs were exposed to a combination of LPS and IFN-γ for 48 hours. This time period of maturation was associated with the development of a mature DC phenotype. In contrast, semi-mature DCs, i.e. DCs matured for 4 hours only, were IDO negative. In co-cultures with allogeneic T cells both types of DCs began to metabolize tryptophan, as determined by decreasing concentrations of tryptophan and increasing concentrations of kynurenines in cell culture supernatants, but mature IDO positive DCs did so at a faster rate (complete consumption of tryptophan within 16 hours of co-culture) than semi-mature DCs. A comparison of the allo-stimulatory capacity of semi-mature IDO negative DCs and mature IDO positive DCs showed that at a high DC/T cell ratio (1:1) IDO positive DCs had a significantly reduced capacity to stimulate allogeneic T cells (median 63% reduction, n=5). The reduction of the allogeneic T cell response induced by IDO positive DCs was reversed upon the addition of the IDO inhibitor methylhydantoin-tryptophan to the co-cultures, suggesting an IDO dependent mechanism. Furthermore, allogeneic T cells exposed to IDO positive DCs hadan increased rate of apoptosis in the activated cell fraction andafter 8 days of co-culture contained a cell fraction (~30%) displaying a CD4+CD25+highFOXP3+ phenotype.These latter cells, when enriched by fluorescent activated cell sorting (FACS), were able to suppress the proliferative response of naive T cells to anti-CD3 mediated stimulation, which indicates the generation of a regulatory T cell population by IDO positive DCs. Together, these findings suggest that the amount of IDO expression and activity by DCs is one feature to govern the type of response of stimulated T cells. Human DCs can be induced to display high levels of IDO expression and activity and, thereby, acquire the ability to effectivley modulate allogeneic T cell responses towards tolerance byeliminating allo-reactive T cells through apoptosis andaugmentation of their regulatory rather than their effector potential.Our current approaches address whether this property can be employed to use DCs for the generation of allo-antigen specific tolerance in the setting of hematopoietic cell transplantation.