Fig 2: PTRF enhances PD-L1 transcription via NF-?B and promotes immune evasion of GBM cells through PD-L1. (A) ChIP-PCR analysis of the ability of NF-?B to bind to the PD-L1 promoter in N9 and TBD0220 cells with or without PTRF overexpression. Results are normalized to IgG. (B) Schematic illustration of the proximal region of the human PD-L1 promoter. (C) N9 and TBD0220 cells (with the expression of empty vector or PTRF) were transfected with a luciferase reporter vector containing WT or mutated NF-?B sequence of PD-L1 promoter. (D) N9 and TBD0220 cells with PTRF or NEAT1 knockdown were transfected with a luciferase reporter vector containing WT or mutated NF-?B sequence of PD-L1 promoter. (E) Flow cytometry analysis of PD-1 binding in TBD0220 cells with or without PTRF overexpression. (F) Quantification of fluorescence intensity in (E). The y axis represents the mean fluorescence intensity of PD-1. (G) Immunofluorescence staining of control and PTRF overexpressing cells. PD-1 (green), Nuclei (blue), scale bar, 20 µm. (H) Quantification of fluorescence intensity in (G). (I) T cell killing assay of TBD0220 cells with or without PTRF overexpression, as well as treatment with NEAT1 knockdown respectively. caspase3/7 cleavage (green), Nuclei (blue), scale bar, 20 µm. **p = 0.01, ***p = 0.001, and ****p = 0.001.

Fig 3: CRAC motifs are critical for the interaction between PD-L1 and cholesterol.(A) Cellular levels of exogenous PD-L1WT, PD-L1F257A, PD-L1F259A, PD-L1R260A, and PD-L1R262A in endogenous PD-L1–knockdown RKO stable cells were analyzed using Western blot. (B) Flow cytometric analysis of membrane PD-L1WT, PD-L1F257A, PD-L1F259A, PD-L1R260A, and PD-L1R262A in endogenous PD-L1–knockdown RKO stable cells. Bar graphs depict the fold change relative to the control; values shown are the means ± SD from four independent experiments (n = 4). Statistical differences were determined with one-way ANOVA. (C) CHX-chase assay showing the degradation of PD-L1WT and mutants. RKO stable cells expressing PD-L1WT, PD-L1F257A, PD-L1F259A, PD-L1R260A, and PD-L1R262A were treated with 50 μM CHX for 2, 4, 6, or 8 hours, and then, PD-L1 was detected by Western blot (shown on the left). The intensity of relative PD-L1 protein (PD-L1 remaining) was quantified using ImageJ analysis (shown on the right). Two independent experiments were performed with similar results. (D) Ubiquitination levels of wild-type PD-L1 (PD-L1WT) and the mutant proteins PD-L1F257A, PD-L1F259A, PD-L1R260A, and PD-L1R262A in HEK293FT cells. Ubiquitination levels were measured with V5 immunoblotting after immunoprecipitation with anti–PD-L1 antibody. Two independent experiments were performed with similar results. (E) Flow cytometry detection of PD-1 binding in RKO stable cells expressing PD-L1WT, PD-L1F257A, PD-L1F259A, PD-L1R260A, or PD-L1R262A. The y axis indicates PD-1 mean fluorescence intensity (MFI). Data are shown as the means ± SD from three independent experiments (n = 3). Statistical differences were determined with one-way ANOVA.

Fig 4: PTRF increases PD-L1 levels and correlates with immunosuppression in GBM. (A) Gene ontology enrichment analysis of genes that are positively associated with PTRF in the CGGA GBM database. (B) Heatmap showing the relationship between PTRF, low tumor purity, and genes regulating immunity in the CGGA GBM database. (C) The correlation between PTRF, CD80, PDCD1, CTLA4, and PD-L1 levels in the CGGA GBM database, measured by Pearson’s correlation test. (D) Western blot analysis of PD-L1 expression in primary GBM cells (N9 and TBD0220; both control and PTRF overexpression cells). (E) Relative mRNA level of PD-L1 in control and PTRF overexpressing cells. (F) Flow cytometry analysis of PD-L1 in control and PTRF overexpressing cells. ****p = 0.001.

Fig 5: PD-L1 CAR NK Cells Kill TFH and Suppress T-Dependent B Cell Responses(A) Control or CAR NK-92 degranulation following a 4-h co-culture with SEB-stimulated tonsillar TFH and CD27+ B cells at an NK:T:B cell ratio of 5:1:2 (n = 5).(B) Percentage lysis (calcein fluorescence in supernatant) of calcein-AM-labeled, SEB-stimulated tonsillar TFH and CD27+ B cells after incubation with control or CAR NK-92 for 4 h at various E:T ratios. Error bars denote standard deviations.(C) TFH and CD27+ B cell PI uptake following a 4-h incubation with control or CAR NK-92 (n = 4).(D) Frequency of proliferating (CTV-) CD27+ B cells, prevalence of CD19+CD27+CD38+ plasmablasts, and total supernatant IgG after 7 days of SEB-stimulated co-culture with tonsillar CD4 T cells that were pre-cultured either without (“No NK”) or with control or CAR NK-92 (n = 3–8). Dotted lines and shaded areas represent measurements in the absence of NK cells. One of 2–3 similar experiments is shown.(E–G) Human cord blood-engrafted NSGS mice injected with pristane were subsequently given weekly infusions of irradiated NK-92 or CAR NK-92 cells (n = 5–7/group, see Method Details). Resulting (E) splenomegaly, (F) splenic CD4 T cell counts, and (G) representative and mean PD-1 expression by human CD4 T cells. Pooled results of 2 independent experiments are shown.Data analyzed via (A, C, E, and F) Student’s t test, (D) 1-way ANOVA with multiple comparisons (comparing each group to “No NK” group”), and (G) Mann-Whitney test.See also Figure S4.