Fig 2: IDO enhances the expression of both CFH isoforms in human GBM. A, Schematic representation of CFH transcript variants reflecting the full-length (CFH) and truncated (FHL-1) sequences. B and C, Pearson correlation analysis of IDO (B) and CD3E (C) with CFH and FHL-1 using the TCGA GBM RNA-seq dataset. D, Left, Western blot analysis of surgically-resected tumor tissue samples from both patients with newly diagnosed GBM and patients with recurrent GBM. Right, Protein expression of full-length and truncated CFH variants in IFN?-stimulated U87 cells. Cells were treated with IFN? and cultured in serum-free medium for 24 to 48 hours. Both cell lysates and cell-culture supernatants were collected. The supernatants were concentrated with ultrafiltration and analyzed by Western blot along with cell lysate samples. One representative result is shown that reflects 4 experimental repeats. E, Time-course analysis of IDO, CFH, and FHL-1 mRNA expression after treatment with IFN? in U87 (top row) and PDX43 (bottom row) GBM cells. The U87 cells were stimulated with 100 ng/mL human IFN? and RNA lysates were extracted followed by qRT-PCR analysis (n = 4). F, Western blot of cell lysates and supernatants collected from the same experimental design as in E. Data from one representative experiment from two experiments are shown. G, Top, Western blot showing IDO protein levels in unmodified (Unmod.) as well as in CRISPR-Cas9 IDO-deleted (IDOKO) U87 cells. Unmod. U87 cells and IDOKO U87 cells were stimulated with 100 ng/mL human IFN? for 24 hours. Protein lysates were collected from both untreated and IFN?-treated cells followed by Western blotting analysis. Bottom, HPLC measurement of Kyn and Trp in Unmod. U87 and IDOKO U87 cells. H, Comparison of IDO, CFH, and FHL-1 mRNA expression induction in Unmod. and IDOKO U87 cells using qRT-PCR (n = 3 per group that reflects 1 representative experiment after two repeats). ***, P < 0.001. All bar graphs represent mean ± SEM.

Fig 3: IDO nonmetabolically increases CFH levels in human GBM. A, Flow chart for the microarray experimental design, data analysis, as well as validation of results. B and C, Real-time RT-PCR confirmation of IDO regulatory effects on CFH expression in cultured U87 (B) or PDX43 GBM (C). Cells were exposed to a variety of conditions including transfection with IDO-specific siRNA (20 nmol/L) or scrambled control siRNA for 16 hours; treatment with or without 100 ng/mL human IFN?; treatment with or without the IDO enzyme inhibitor, BGB-5777 (1 µmol/L); or IFN? plus BGB-5777 for 24 hours. qRT-PCR was performed (n = 3 reflecting one representative experiment from four experimental repeats). D, qRT-PCR analysis of IDO and CFH mRNA expression on IFN?-stimulated U87 cells treated with CFH siRNA or scrambled control siRNA. Same experimental conditions as in B (n = 3/group reflecting one representative experiment from three experimental repeats). E, mRNA expression levels for human IDO, CFH, and CD3e were quantified and compared among intracranial PDX43 and patient-resected newly-diagnosed or recurrent intracranial human GBM (n = 4–9/group). Intracranially-engrafted (ic.) PDX43 was isolated from humanized mice with or without treatment of CD4+ and CD8+ T-cell depleting antibodies between 14 and 21 days post intracranial injection. ***, P < 0.001. All bar graphs represent mean ± SEM.

Fig 4: Systemic and local complement factor H levels and the relationship with other immunosuppressive factors in patient-resected GBM. A and B, Quantification of protein levels of full-length and truncated CFH in patient plasma samples by mass spectrometry. C, Pearson correlation analysis for CFH mRNA with PD-L1, PD-L2, PD-1, CTLA-4, STAT3, CD39, BTLA, LAG3, FOXP3, and FGL2 in GBM. Each small circle in the plot represents the expression in a single patient. D, Canonical correlation analysis of CFH with major tumor immune cell types. The signature genes of each type of immune cells are defined as: CD8+ T cell (CD3e, CD8a), Treg (CD3e, CD4, CD25, FoxP3), MDSC (CD14, CD11b, CD33, Arg1), TAM (CD14, HLA-DR, CD312, CD115, CD163, CD204, CD301, CD206), and neutrophil (CD11b, CD16, CD66b, ELANE). E, Schematic presentation of a hypothesis based on the findings reported here and elsewhere. Standard of care treatment radiation (RT) and TMZ enhances inflammatory mechanisms that alter the immune-tolerant (cold) GBM microenvironment into more inflamed (hot) conditions which is partially caused by tumor-infiltrating IFN?+ CD8+ T cells. The IFN? acts on human GBM cells to induce IDO expression, which in turn, enhances CFH expression levels through a nonenzymatic mechanism. CFH acts on complement receptors in an autocrine and paracrine manner; the latter of which elicits CCL2 expression in TAMs. The TAMs then facilitate Treg and additional monocyte recruitment into the GBM which reinforces the immunosuppressive microenvironment.

Fig 5: IDO and CFH mRNA levels positively correlate with T-cell infiltration in patient-resected GBM. A, mRNA expression levels for IDO and CFH in grade II (green; n = 226), grade III (blue; n = 249), and grade IV (GBM; red; n = 172) glioma of the RNA Hi-Seq. Illumina dataset as analyzed in TCGA. Horizontal lines in the scatter plots represent mean ± SEM. B, Pearson correlation analysis for IDO and CFH mRNA levels within GBM and all-grade glioma. C, Kaplan–Meier survival analysis of grade II (left), grade III (center), and grade IV (GBM, right) patients with glioma stratified by low CFH (blue) and high CFH (red) expression levels. D, Kaplan–Meier analysis of GBM recurrence. Recurrent GBM samples were identified from the ‘days to tumor recurrence’ section listed in the TCGA GBM clinical dataset. CFH mRNA expression levels were extracted from the Affymetrix U133a microarray dataset. E, CFH mRNA levels were compared among grade II (IDHwt; blue circle and IDHmut; red square), grade III (IDHwt; green triangle and IDHmut; purple circle), as well as grade IV (IDHwt; orange circle and IDHmut; black triangle) glioma. F, CFH DNA methylation analysis at two distinct genomic loci, cg06377993 and cg23557926 in grade II (IDHwt; blue circle and IDHmut; red square), grade III (IDHwt; green triangle and IDHmut; purple circle), and grade IV (IDHwt; orange circle and IDHmut; black triangle) glioma. G, Expression of CFH and IFN? mRNA levels in patient-resected GBM tissue samples as categorized by CD3E and CD8A expression levels while accessing the TCGA GBM RNA-seq dataset. H, Detection of CFH mRNA in the human GBM cell T-cell coculture system in vitro. CD3+ human T cells were isolated under positive selection from PBMCs of patients with GBM. CFH mRNA levels were analyzed in U87 GBM cells cocultured with either naïve or activated T cells or conditioned medium from activated T cells in the presence or absence of IFN?-neutralizing antibodies. Data were compiled from 3 independent experiments. I,In vitro expression analysis of human CFH mRNA in different GBM cells with or without the addition of human IFN?. Data represent pooled data from 4 independent experiments. **, P < 0.01; ***, P < 0.001. All bar graphs represent mean ± SEM.