The survival rate of patients with glioma has not significantly increased in recent years despite aggressive treatment and advances in immunotherapy. The limited response to treatments is partially attributed to the immunosuppressive tumor microenvironment, in which regulatory T cells (Treg) play a pivotal role in immunologic tolerance. In this study, we investigated the impact of complement factor H (FH) on Tregs within the glioma microenvironment and found that FH is an ICOS ligand. The binding of FH to this immune checkpoint molecule promoted the survival and function of Tregs and induced the secretion of TGFβ and IL10 while suppressing T-cell proliferation. We further demonstrated that cancer cells in human and mouse gliomas directly produce FH. Database investigations revealed that upregulation of FH expression was associated with the presence of Tregs and correlated with worse prognosis for patients with glioma. We confirmed the effect of FH on glioma development in a mouse model, in which FH knockdown was associated with a decrease in the number of ICOS+ Tregs and demonstrated a tendency of prolonged survival (P = 0.064). Because the accumulation of Tregs represents a promising prognostic and therapeutic target, evaluating FH expression should be considered when assessing the effectiveness of and resistance to immunotherapies against glioma.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.
This work is licensed under a Creative Commons Attribution 4.0 International License .
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Supplementary data

Supplementary Figure 1

Supplementary figure 1. FH binds to T-cells via ICOS

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Supplementary Figure 2

Supplementary figure 2. FH in Ntv-a mouse model (A) Mouse FH binds to mouse derived primary Tregs. Tregs were incubated for 2 h at 4oC with fluorescently labeled 25 or 100 μg/mL FH. The binding was detected using flow cytometry. (B) Western blot detecting FH in supernatants of tumor cells isolated from Ntv-a mice. FH was partially depleted with antibody against mouse FH bound to Dynabeads. (C) FH-rendered increase in survival of mouse Tregs. The cells were incubated with tumor cell derived supernatant and FH-depleted supernatant. After 7 days viability was assessed by Annexin V and Via-Probe staining. (D) FH was successfully knockdown with shFH. DF1 cells were transfected with three different shFH constructs and Gl2 shRNA. The knockdown of FH in NIH3T3 cells, transfected with supernatants from the DF1-RCAS-shFH/DF-1-RCAS-shGl2 cells was detected with goat anti-FH antiserum by western blot. (E) Control staining of mouse tumor sections. Samples were incubated with goat anti-FH antibody, goat IgG isotype control, and anti-goat IgG secondary antibody or only secondary antibody. Nucleus was stained with DAPI. Data are means ± SD of (A) n = 4, (C) n = 3 independent experiments. Representative blot (D) of n = 3 and picture (E) of n = 3 independent experiments. Statistical tests: two- Kruskal-Wallis with Dunn´s multiple comparison test (A, C). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns -, nonsignificant; CTRL, control.

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Supplementary Figure 3

Supplementary figure 3. Direct effect of FH on glioma cells Proliferation of PIGPC cells treated with medium only, 25- or 100-μg/mL FH (A) and H4 mock or FH-transfected (B) was analyzed after 24, 48, 72 and 96 hours using CyQUANT assay. Data obtained at 24 hours was used for the normalization. (C) Correlation between FH and Ki67 gene expression in tumor cells from glioma patients, obtained from GEO database. Survival of PIGPC cells pretreated with 100 μg/mL FH (D-F) and H4 mock or FH-transfected (G-I) rendered apoptotic by treatment with 0.75 uM staurosporine for 24 h. Viability was assessed by Annexin V and Via-Probe staining. Data are means ± SD of (D, E, F) n = 4, (A, B, G, H, I) n = 3 independent experiments. Statistical tests: Two-way ANOVA with Bonferroni's multiple comparisons test (A, B), one-way ANOVA with Tukey´s multiple comparison test (D-I) Spearman’s rho correlation (C). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, nonsignificant; CTRL, control.

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Supplementary Figure 4

Supplementary figure 4. Staining control for human samples and additional information about patients (A) Histology and (B) clinical parameters of glioma patients. MGMT-methylation status; TMZ-temozolomide; Adj-adjusted. WT – wild type.

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Supplementary Figure 5

Supplementary figure 5. FH, ICOSL and ICOS dependence on glioma patient survival. The survival data of n = 509 patients from TCGA provisional dataset brain lower-grade glioma, analyzed with cBioPortal. Statistical tests: Logrank Test.

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Supplementary Figure 6

Supplementary figure 6. FH expression in glioma correlates with pro-tumorigenic markers

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