Fig. 2

Flow cytometric monitoring of System L-dependent kynurenine uptake. a Flow cytometric evaluation of kynurenine uptake in IL-2 maintained CTLs. Data acquired using 405 nm excitation (violet laser) and band pass filter 450 ± 50 on BD LSRII (Fortessa). The left panels show a dot plot of fluorescence (emission 450 nm) of CTLs against time. Data acquired pre (120 s) and post (+700 s) addition of 200 μM kynurenine (indicated by red arrow). The centre panel shows the same data plotted as a geometric mean trace (FlowJo software). The right panel shows overlaid histograms of the data from CTLs in the presence or absence of kynurenine (200 μM) for 4 mins (37 °C). b The graphs show the number of ³H-kynurenine molecules transported per cell per minute (left), or mean fluorescence intensity (MFI, right) of IL-2 maintained CTLs when incubated with increasing kynurenine concentrations. K_m values, the concentration of kynurenine that gives half maximal uptake, were calculated using standard Michaelis–Menton equation (GraphPad Prism software). c 450 nm fluorescence in IL-2 maintained CTLs treated with kynurenine, as in a, in the presence or absence of the System L inhibitor, BCH (10 mM). d 450 nm fluorescence emission of 36 h CD3/CD28 stimulated CD8⁺ T cells (left) or IL-2 maintained CTLs (right) treated with kynurenine in the presence or absence of BCH (10 mM), Leu (5 mM) or Lys (5 mM) at (37 °C) for 4 min. e 450 nm fluorescence emission of IL-2 maintained CTLs treated with kynurenine in the presence or absence of BCH (10 mM), at (37 °C); or treated with kynurenine at (4 °C) for 4 min. f The histograms show 450 nm fluorescence in CTLs treated with kynurenine for 4 mins ± BCH (10 mM). Cells were analysed immediately (live; top panel) or after fixation (fixed; bottom panel) using 1% paraformaldehyde. MFIs are indicated in the histograms. Data shown are representative of 6 (CTL) or 4 (TCR) experiments