Fig 1: PD-1 as a signaling receptor activates NF?B transcription factor in BTICs.(A) Schematic diagram of site-directed mutagenesis in two signaling motifs (ITIM, Y223F; ITSM, Y248F) of human PD-1 gene. (B) Representative flow cytometry plots of PD-1 expression in BT073 transfected with OE vectors coding wild-type (WT), 223, 248, or 223/248 mutant (Mut) version of PD-1. (C and D) ATP proliferation assay of human BT048 and BT073 cells OE wild-type or mutant versions of PD-1 compared with OE control. ns, not significant. (E) Heatmap of differentially expressed genes between PDCD1–OE cells and OE control of three human BTIC lines. Experiment was conducted once. (F) Volcano plot shows all data points that meet the false discovery rate (FDR) cutoff of 5% and 1.5-fold change criteria. (G) Validation of mRNA expression of TNFRSF19 and IKBKB genes by RT-qPCR in another culture set of BT048 and BT073 cells. Fold changes were calculated relative to PD-1 expression in OE control and normalized to GAPDH expression. (H) The phosphorylation of IKKa/ß, SHP-2, and NF?B (p65) was detected in the cell lysates of four human BTIC lines by Western blot. Actin was used as a loading control. Luminescence ATP proliferation test of PDCD1-OE BT048 and BT073 cells compared to OE controls after 72 hours of treatment with (I) SHP-2 inhibitor RMC4550 (3 Mµ) or (J) IKKa/ß inhibitor BMS-345541 (1 Mµ). DMSO, dimethyl sulfoxide. (K) After PD-1 immunoprecipitation of cell lysates and SDS–polyacrylamide gel electrophoresis, immunoblot was performed with antibodies for p–SHP-2, SHP-2, p-IKKa/ß, IKKß, and PD-1. Means were compared by unpaired (two-tailed) t test when comparing two groups. For more than two groups, one-way ANOVA with Tukey’s post hoc was used: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are represented as means ± SEM. See also fig. S5.
Fig 2: Tumor-intrinsic PD-1 promotes BTIC growth in culture.(A) Confirmation of PDCD1 down-regulation or up-regulation in human BTIC lines at mRNA levels by RT-qPCR. Fold changes were calculated relative to PD-1 expression in respective vector controls and normalized to GAPDH expression. (B) Flow cytometry analysis of PD-1, PD-L1, and PD-L2 expression after knocking down or OE PD-1 in BTICs. (C and D) Representative bright-field microscopy images of 72- to 96-hour outcomes of tumor spheres in PD-1 knockdown or overexpression versus respective vector controls of two human (BT048 and BT073) and mouse (mBT0309) BTIC lines. (E and F) Quantification of tumor spheres in PD-1 down-regulated or PD-1 OE versus respective vector controls. (G and H) ATP proliferation assay of human and mouse BTIC with PD-1 down-regulation or up-regulation. (I and J) Representative plots and bar plots of the proliferation of human BTIC lines by measuring incorporation of EdU into DNA following 24-hour treatment. AF488, Alexa Fluor 488; RLU, relative light unit. Data are representative of two to three separate experiments. Means were compared to respective vector controls by unpaired (two-tailed) t test when comparing two groups. For more than two groups, one-way analysis of variance (ANOVA) with Tukey’s post hoc was used: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are represented as means ± SEM. See also fig. S4.
Fig 3: The expression of PD-1 on BTICs in resected clinical GBM specimens in situ.Representative images of immunofluorescence staining and corresponding 3D reconstruction of images of (A and B) CD45 and PD-1, (C and D) PD-1 and BTIC marker SOX2, and (E and F) PD-1 and BTIC marker nestin in sections of human GBM specimens resected from three patients (1085, 110907, and 101029). Representative immunofluorescence images of human normal brains stained for (G) PD-1 and SOX2 or (H) PD-1 and nestin. (I) Quantification of immunofluorescence staining of PD-1+ SOX2+ or PD-1+ nestin+ cells within the tumor microenvironment. Quantification was performed in three to four fields of view (FOVs) of each patient. Each circle is of a different patient with GBM, and the color of the dots in both panels match to the same subject. Green, 026-1; purple, 101220; orange, 110907; blue, 100819; red, 110512; black, 101029; yellow, 1085. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Data are represented as means ± SEM. See also fig. S1 and table S1.
Fig 4: Coexpression of PD-1, NF?B activation, and proliferation marker in BTICs in GBM specimens.(A) Representative immunofluorescence laser confocal microscopy images and quantification of PD-1, SOX2, and p65 in sections of human GBM specimens resected from three patients (110907, 101220, and 100819). Quantification was performed in three to four FOVs of each patient. Nuclear translocation (yellow arrows) of p65 in BTICs is shown on the magnified images. Each dot in the bar graphs corresponds to the number of cells per FOV. (B) Representative images of immunofluorescence staining of PD-1, SOX2, and proliferative marker Ki67 in sections of human GBM specimens from three patients, with some triple-positive cells indicated by yellow arrowheads. Nuclei were counterstained with DAPI. Means were compared by unpaired (one-tailed) t test when comparing two groups. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 5: Diagram of PD-1 expression on BTICs and signaling through NF?B.PD-1 is expressed on BTICs in resected clinical GBM specimens in situ and patient-derived BTICs in culture. Tumor-promoting effects of PD-1 in BTICs did not require interaction with PD-L1; thus, the therapeutic antibodies were unable to overcome the growth advantage of PD-1 in BTICs. Mice with intracranial Pdcd1 over- or underexpressing BTICs had shorter or longer survival, respectively. Mechanistically, phosphorylation of ITIM and ITSM motifs within the cytoplasmic tail of PD-1 recruited SHP-2 phosphatase and activated the NF?B pathway through IKKa/ß in BTICs.
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