Fig 2: NMR characterizations of PD-L1-TC in bicelles.(A) The 2D 1H, 15N-TROSY-HSQC spectrum of PD-L1-TC samples in DMPC/DH6PC bicelles recorded at 310 K on a Bruker 600-MHz spectrometer. ppm, parts per million. (B) Cartoon representation of the 15 lowest-energy structures of PD-L1-TC in DMPC/DH6PC bicelles. The calculated helical region contains amino acids 238 to 261 (blue) with a length of 38.6 Å, covered by the predicted transmembrane region (amino acids 239 to 259, green) from UniProt (right). (C and D) Measurement of membrane immersion depth of PD-L1-TC in DMPC/DH6PC bicelles using the water-soluble probe Gd-DOTA. 1H, 15N-TROSY NMR spectra of PD-L1-TC samples in the absence (red) and presence (black) of 15 mM Gd-DOTA; both spectra were acquired at 310 K on a Bruker 600-MHz spectrometer (C). PRE effects (I/I0) versus [Gd-DOTA] for N236 and L248 were plotted at different concentrations of Gd-DOTA (D). I, peak intensity in the presence of Gd-DOTA; I0, peak intensity in the absence of Gd-DOTA. (E) Assessment of PD-L1-TC in DMPC/DH6PC bicelles from the PRE effects of hydrophilic Gd-DOTA and lipophilic16-DSA paramagnetic probes. Peak intensity reductions induced by 16-DSA (2 mM) and Gd-DOTA (15 mM) relative to a reference spectrum are shown as bar graphs in orange and green, respectively. Measurements were carried out for PD-L1-TC in DMPC/DH6PC bicelles at an 1H frequency of 700 MHz (pH 6.5) and 310 K. (F) Membrane partitioning of PD-L1-TC as determined by PRE effects. The position of the PD-L1-TC structure is shown along the membrane axis of the lipid bilayer. The transmembrane region (amino acids 239 to 261) is highlighted in gray, the juxtamembrane region (amino acids 262 to 272) is highlighted in light blue, and the cytoplasmic region (amino acids 273 to 290) is highlighted in pink, with residues 236, 251, and 261 colored green, red, and purple, respectively. The complete PREamp plot from Gd-DOTA titrations is shown on the right.

Fig 3: Anti-tumor activity of vorinostat, anti-PD-1 and anti-CTLA-4 on established mouse breast cancer allografts(A) 4T1 cells (1 x 106) were s.c. injected into BALB/C mice. When established tumors were palpable, mice were treated with vorinostat (100 mg/kg i.p.), anti-PD-1 (10 mg/Kg i.p.) combined with anti-CTLA-4 (10mg/Kg i.p.) or a combination of the three drugs as described in the Material and Methods. Relative tumor volume curves for 4T1 allograft; measurements are shown as mean ± SEM tumor volume (n = 8). (B) Tumor volume averages from each group at day 0 and day 25 (end of treatment) were compared and presented as percentages of vehicle. (C) Effect of vorinostat and/or anti-PD1 + anti-CTLA-4 treatments on the survival of 4T1 allograft mice. (D) Mouse PD-L1 mRNA was measured in FFPE 4T1 tumor samples by RNAscope assay. Hybridization signals were amplified and visualized with RNAscope 2.0 HD detection kit. Data are presented as the mean ± SEM. (E) Murine PD-L1 RNAscope images were captured under a bright field at 40x magnification. One representative image for each treatment is shown. Positive signals showed as brown punctuate dots were analyzed by scoring with ImageJ software. Statistical significance is indicated by p-values as * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; Ns: non significant.

Fig 4: Characterization of cholesterol binding sites in PD-L1-TC.(A) Superimposed 2D 1H, 15N-TROSY-HSQC spectra of 0.2 mM 15N-labeled PD-L1-TC were acquired at 700 MHz (1H frequency) and 310 K in the presence of cholesterol at 0 mM (red), 0.5 mM (orange), 1.0 mM (green), 1.5 mM (blue), 2.0 mM (purple), or 2.5 mM (black). (B) Enlarged view of four regions in the plot displayed in (A), showing specific chemical shift perturbations with cholesterol titration of the two CRAC motifs (CRAC1: F257 and R260; CRAC2: F259 and R262) (right). (C) NMR chemical shift changes of PD-L1-TC after the addition of 1.5 mM cholesterol in DMPC/DH6PC bicelles. The plot shows chemical shift changes between the spectra of PD-L1-TC with or without cholesterol. (D) Chemical structures of CNO (left) and DOXYL-CH (right) are shown on in the topmost panel. (E) PRE (I/I0) effects of different nitroxide-labeled cholesterols (DOXYL-CH and CNO) on PD-L1-TC in DMPC/DH6PC bicelles. Specific broadening of 1H-15N correlation peaks by 2.4 mM CNO and DOXYL-CH and intensity recovery of the broadened peaks with the addition of ascorbic acid are shown in the middle and bottom panels, respectively. (F) PRE (I/I0) analysis of CNO and DOXYL-CH. The addition of 2.4 mM CNO showed obvious PRE effects at L252 to A254, whereas addition of 2.4 mM DOXYL-CH caused the most notable PRE effects at R260 to R262. I, peak intensity in the presence of the paramagnetic agent; I0, peak intensity in the absence of the paramagnetic agent. (G) Comparison of (1-PRE) caused by DOXYL-CH (left) and CNO (right) with simulated binding curves. For the PRE data, PD-L1-TC NMR samples were titrated with 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 2.4 mM DOXYL-CH and CNO, respectively. The simulated binding curves with n = 1 (no cooperativity, dotted black line) and n = 2 (positive cooperativity, dotted red line) are shown in each diagram, respectively.

Fig 5: Schematic diagram depicting the effect of cholesterol on PD-L1 stability.Cholesterol can directly bind to the transmembrane domain of PD-L1 at two CRAC motifs, stabilizing PD-L1 on the cell membrane. Reducing cholesterol levels with a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (simvastatin) or cholesterol depletion reagent (MCD) decreases levels of membrane-bound PD-L1 by promoting PD-L1 ubiquitination and degradation. Mutations in the CRAC motifs have similar effects, reducing levels of PD-L1 on the cell surface. The magnified region shows key residues (CRAC1: F257/R260; CRAC2: F259/R262) in the two cholesterol-binding sites of PD-L1-TC.