Figure 4: PD-1 induces lipolysis and utilization of endogenous fatty acids for β-oxidation.
(a) The amounts of n3DPA;22:5n3 and glycerol-3-phosphate (G3P) in the cells and the amounts of DHA;22:6n3 and 3-hyroxybutyrate (BHBA) in the culture supernatants of the indicated culture conditions were analysed and values were plotted in whisker boxes. Values of TCC and TCC+PD1 cells were compared with unstimulated (UT) cells (♦P<0.05, analysis of variance (ANOVA); n=5) and values of TCC+PD1 were compared with TCC cells (*P<0.05, Welch t-test; n=5). (b) Purified human T cells were cultured under the indicated conditions. Cell lysates were prepared at the indicated time points and expression of FASN, ATGL and β-actin were assessed by SDS–PAGE and immunoblot. Results are representative of three experiments. (c) ATGL 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 (UT) cells (defined as 1) is shown. Expression levels of ATGL mRNA in TCC+PD1-stimulated cells were compared with TCC-stimulated cells (*P<0.05, Student’s t-test) and expression levels in TCC and TCC+PD1-stimulated cells were compared with unstimulated (UT) cells (♦P<0.05, ANOVA). Results are mean±s.e.m. of three experiments. (d) T cells were cultured with [9,10-3H]palmitate for 48 h, over which time the radiolabelled fatty acid was used to generate cellular lipids. Subsequently, cells were washed to remove unincorporated label and were cultured with beads conjugated with aCD3/CD28/IgG or with beads conjugated with aCD3/CD28/PD-L1-Ig and the amount of β-oxidation of labelled fatty acids generated from endogenous lipid was determined each day. Values of TCC and TCC+PD1 cells were compared with unstimulated (UT) cells (♦P<0.05, ANOVA; n=3) and values of TCC+PD1 were compared with TCC cells (*P<0.05, Student’s t-test; n=3).
