Figure 1: PD-1 inhibits transport and utilization of glucose during T-cell activation.

CD4⁺ primary human T cells were either left unstimulated (UT) or were incubated with magnetic beads conjugated with αCD3/αCD28/IgG (T cells co-stimulated (T_CC)) or magnetic beads conjugated with αCD3/αCD28/PD-L1-Ig fusion protein (T cells co-stimulated+PD-1 (T_CC+PD1)). (a) IFN-γ production was assessed by ELISA (*P<0.05 T_CC+PD1 versus T_CC; n=3 experiments; Student’s t-test). (b,c) Expression of Glut1 after culture under the indicated conditions and time intervals was examined by flow cytometry, and glucose uptake was examined by 2-{¹⁴C(U)}-deoxy-D-glucose incorporation. At each time point glucose uptake in T_CC+PD1-stimulated cells was compared with T_CC-stimulated cells (*P<0.05, T_CC+PD1 versus T_CC; n=3 experiments; Student’s t-test). (d) Analysis of key metabolites involved in glycolysis was performed in cells and culture supernatants as described in Methods. The amounts of the indicated metabolites in unstimulated, T_CC and T_CC+PD1 cells were plotted in whisker boxes. The lower and upper sides of the box indicate the first and third quartile, respectively. The horizontal line inside the box indicates the median value, whereas the lower and upper bars indicate the minimum and maximum of distribution, respectively; (+) mean value; (o) extreme data point. Results of five measurements generated from five independent experiments (*P<0.05, T_CC+PD1 versus T_CC, Welch’s t-test; ^♦P<0.05, T_CC versus UT and T_CC+PD1 versus UT, analysis of variance). (e) HK2 mRNA was analysed by real-time quantitative PCR and relative expression of mRNA of each time point and culture condition over the levels expressed in unstimulated cells (defined as 1) is shown. Analysis was performed over prolonged time of culture (120 h) to investigate whether quantitative difference versus altered kinetics of HK2 expression was induced by PD-1 (*P<0.05, T_CC+PD1 versus T_CC, Student’s t-test; n=3 experiments).