Human Monocyte-Derived Dendritic Cells Are Tolerogenic through Both IDO1 and IDO2

Abstract 4298

Indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like (IDO2) are enzymes involved in the tryptophan catabolism along the kynurenine pathway. While it is established that IDO1-expressing dendritic cells (DCs) contribute to tolerance in a number of biological settings, little is known about the expression and function of IDO2 in DCs. Human DCs can be generated in vitro to obtain immunogenic antigen-presenting cells (APC), used as cellular vaccines. In the clinical setting, DCs are commonly matured with a cytokine cocktail (CC) which includes TNF-a, IL-1b, IL-6 and PGE₂. In particular, PGE₂ enhances APC function of DCs by increasing IL-12 production and facilitating DC migration to lymph nodes. However, PGE₂ is also a strong IDO1 inducer, which by this route can also limit the anti-tumor activity of DC-based immunotherapies. Thus, understanding the roles of IDO1 and IDO2 in DCs may impact the development of vaccines or DC-based immunotherapies. In the present study, we fully characterized IDO1 and IDO2 expression and function in human monocyte-derived dendritic cells (Mo-DCs). Mo-DCs were generated from purified CD14⁺ monocytes after culture with GM-CSF and IL-4 and then matured with CD40L, LPS alone, LPS plus IFN-g and the CC.

We observed that immature Mo-DCs had little if any expression of both IDO1 and IDO2, whereas mature Mo-DCs exhibited upregulation of both enzymes. Among the different maturation stimuli, CC was the most effective in upregulating IDO1 and IDO2, both at the message and protein levels. This effect was associated also with the highest kynurenine production. By means of IDO1 and IDO2 expression, mature Mo-DCs were inhibited in stimulating allogeneic T cell proliferation and generated a population of CD4⁺CD25⁺FOXP3⁺ T_regs which highly suppressed allogeneic and autologous T-cell proliferation. On the basis of evidence that IDO1 is preferentially inhibited by the L-isoform of 1 methyl-tryptophan (1-MT) and IDO2 by the D-isoform, we performed functional enzyme tests in presence of both isoforms. Notably, both isoforms exhibited inhibitory effects, although we observed a stronger effect of L-1-MT than with D-1-MT suggesting a greater contribution of IDO1 than IDO2. These results offer direct evidence that Mo-DCs express functional IDO1 and IDO2 proteins. During the maturation phase, Mo-DCs enhance their tolerogenic qualities, and in particular the capacity to induce T_regs, through the upregulation of both IDO1 and IDO2. Beside the critical role of IDO1 in enhancing the immunosuppressive capacity of DCs, we show, for the first time, that IDO2 is involved also. Our findings imply that, from a clinical standpoint, to improve the efficacy of DC-based vaccines mature DCs should be combined with molecules that can inhibit the activity of both IDO1 and IDO2.