Fig 2: Palmitoylation promotes tumor-intrinsic PD-1 signaling. (A) Interaction between endogenous PD-1 and S6 in tumor cells by co-IP. (B–D) The interactions between PD-1 and S6/eIF4E were significantly decreased by depleting DHHC9 (B), 2-BP treatment (C) and mutating Cys192 (D), as shown by co-IP. (E) Modulating the expression level of PD-1 by 2-BP and PalmB impacts the phosphorylation level of the mTOR effectors eIF4E (left panels) and S6 (right panels). (F) Modulating the expression level of PD-1 by 2-BP and PB in a dose-dependent manner correlates with the phosphorylation of the mTOR effectors eIF4E (left panel) and S6 (right panel). (G) Overexpression of PD-1 other than its mutant C192S increased the phosphorylation level of eIF4E and S6 in cancer cells. (H) Phosphorylation of eIF4E and expression of PD-1 in RKO cells treated with siRNAs specific for DHHC9. Statistics shown on the right, *** P < 0.001, ANOVA test. n = 3 independent experiments. (I) The phosphorylation of eIF4E and S6 is increased in HepG2 cells overexpressing DHHC9. Statistics shown on the right, *** P < 0.001, ** P < 0.01, ANOVA test. n = 3 independent experiments. (J) The phosphorylation of eIF4E and S6 is not affected by PD-L1 silencing or PD-L1 overexpression.

Fig 3: Obesity was associated with repression of immune checkpoints. (A) The expression of various immune checkpoints in obesity and normal groups. (B) The expression of LAG3 and PD-1 in healthy bowel tissues, colon cancer derived from normal (non-obesity) patients and obesity patients was confirmed by immunohistochemistry. (C) The association of drug sensitiveness and gene expression (LAG3 and PD-1) analyzed by Genomics of Drug Sensitivity in Cancer (GDSC) database. (D) The correlation matrix of each immune checkpoint in obesity and normal group. (*p<0.05, **p<0.01).

Fig 4: Screening results of cross-reactive antigens against anti-PD-1 antibody using CF-PA2Vtech (a 1536-well plate version). (A) The result images of the first screening using anti-PD-1 antibody by 1536-well plate based CF-PA2Vtech. Ten spots shown as black dots were positive. A blue ellipse indicates the spots of human PD-1 protein as a positive control. (B) The dot-blotting graph from scan data of the first screening using anti-PD-1 antibody. Signal intensities of each array spot were plotted. On the graph, four spots (red colour characters) were selected as positive spots. (C) The result images of second screening using anti-PD-1 antibody. The proteins in positive wells found in the first screening were re-arrayed in duplicate for individual testing on the array. Four proteins, shown as clone 1 to 4, were recognized by the anti-PD-1 antibody (red ellipses). Clone 4 was the PD-1 protein from the protein array. Green ellipse was TRIM21, which cross-reacts with anti-IgG antibody. (D) The dot-blotting graph from scan data of the second screening using anti-PD-1 antibody. Average signal intensities of dual spots on an array image were plotted. On the graph, four spots showed higher signal intensities. The two spots (blue colour character, 05–15/16, 27–33/34) were the positive control, and one spot (green colour, 07–17/18) was TRIM21 reacting with anti-IgG antibody. The remaining three red colour spots (09–39/40, 13–09/10, 13–35/36, 15–23/34) and PD-1 spot (15–23/24) were determined as cross-reactive proteins by anti-PD-1 antibody we used.

Fig 5: Outcome of PD-1-PD-L1 Engagement at T Cell-APC Interface(A) Jurkat-PD-1OST cells were stimulated with Raji cells that have been preincubated in the absence (-) or presence (+) of 200 ng/mL SEE and lysed 2 min after the initiation of cell-cell contact. Immunoblot analysis of equal amounts of lysates from the specified conditions probed with antibody to phosphorylated proteins (Anti-p-Tyr) or with phospho-tyrosine-specific antibodies directed against SLP76 pY128, ZAP70 pY493, LAT pY171 or VAV1 (loading control). Left margin, molecular size in kilodaltons (kDa). Data are representative of three independent experiments.(B) Expression of CD3, CD28, PD-1, PD-L1, PD-L2, BTLA, and HVEM at the surface of Jurkat cells and Jurkat-PD-1OST cells, analyzed using flow cytometry.(C) Expression of HLA-DR, PD-L1, PD-L2, CD80, CD86, and HVEM at the surface of Raji cells and Raji-PD-L1 cells, analyzed using flow cytometry. In (B) and (C), gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments.(D) IL-2 production by Jurkat and Jurkat-PD-1OST cells stimulated for 24 h with either Raji or Raji-PD-L1 cells in the absence (0) or presence of the specified amounts of SEE. Data are representative of three independent experiments, and mean and SEM are shown.(E) Jurkat-PD-1OST cells stimulated at 37°C with Raji or Raji-PD-L1 cells preincubated in the absence (–) or presence (+) of 200 ng/mL SEE and lysed 2 and 5 min after the initiation of cell-cell contact. Immunoblot analysis of equal amounts of lysates from the specified conditions subjected to affinity purification on Strep-Tactin-Sepharose beads and probed with antibody to phosphorylated proteins (Anti-p-Tyr). Also shown are loading controls probed with anti-PD-1 antibody. Left margin, molecular size in kilodaltons (kDa). Data are representative of two independent experiments.