Peripheral blood lymphocytes were labeled with CFSE and cultured in preconditioned supernatants from Hela cells in 96 well flat-bottomed plates, pre-coated with PBS or with antibodies against human CD3 and CD28. After 4d the cells were harvested, stained with anti human CD3-APC and acquired on the flow cytometer (FACScalibur), gating on propidium iodide negative (live) cells and analyzed using FlowJo software. A. Representative dot plots are shown from cells cultured in conditioned supernatant from WT, WT + IFN-γ, GFP or IDO Hela cells, with CFSE against CD3-APC and T cell proliferation is shown by dilution of CFSE. B. T cell proliferation in pre-conditioned supernatant can be restored to normal levels by adding 25μM fresh L-tryptophan to the T cell cultures. Data are shown as percentage of CD3+ T cells that have undergone proliferation and are the means of duplicate wells from two independent experiments combined ± SE. The data shown are representative from at least three experiments. Statistical significance <0.05 as assessed using Student’s T-test is indicated by *.