Fig. 4

Indoleamine 2,3-dioxygenase (IDO) activity and effects. a The histograms indicate the median values (range) of kynurenine concentrations (the white panels indicate the use of T cells from healthy mucosa, the light grey panels indicate the use of T cells from non-inflamed mucosa, and the dark grey panels indicate the use of T cells from inflamed mucosa of patients with Crohn’s disease) of at least three separate experiments performed in triplicate in each experimental condition. b Immunoblotting of mesenchymal stem cells (MSCs) transfected with both the siRNA targeting (MSC siRNA) or non-targeting (MSC control) IDO with the mouse monoclonal anti-IDO antibody. The protein levels were measured by scanning densitometry as band area and band intensity, expressed as arbitrary units, and normalized toward β-actin levels. In the upper part of the panel, a representative case is shown, and in the lower part, the values are given. c-h The histograms indicate the median values (range) of viable T cells, their dead rate, and proliferative response in the presence of 1-MT (c-e) and transfected MSC (f-h) and refer to at least three separate experiments performed in triplicate in each experimental condition. White panels indicate the use of T cells from healthy mucosa, the light grey panels indicate the use of T cells from non-inflamed mucosa, and the dark grey panels indicate the use of T cells from inflamed mucosa of patients with Crohn’s disease. For comments, see the text. At the preliminary analysis of variance, both the 1-MT and the use of MSC transfected with siRNA targeting IDO affected the variability of our experimental parameters (P < 0.0001 in all cases). The reported P values derive from the non-parametric tests applied to the direct comparisons. ctr-TCL control T-cell line, MSC-T T-cell lines obtained in the presence of mesenchymal stem cell, 1-MT 1-methyl-DL-tryptophan, SI stimulation index, siIDO siRNA small interfering RNA