The immune checkpoint VISTA exhibits high expression levels in human gliomas and associates with a poor prognosis

Abstract

In human gliomas, anti-tumor T cell responses are inhibited through induction of local and systemic immunosuppression. Immune checkpoint blockade is proving to be a success in several types of cancers. However, many studies reported that the treatment of glioblastoma patients with anti-CTLA-4 or anti-PD-1 has no survival benefit compared to standard chemotherapy. This study aimed to investigate the expression and role of VISTA, a newly described immune checkpoint regulator, in human gliomas. mRNA expression was assessed in a total of 87 samples from glioma patients. 57 glioma tissues were taken at different grades. 20 peripheral blood mononuclear cells (PBMC) samples were taken before surgery and ten after surgery, all from the same set of patients. As for the control, ten specimens of PBMC were taken from healthy donors. Protein expression using immunohistochemistry was performed for 30 patients. The Cancer Genome Atlas (TCGA) data set, was also used to investigate VISTA expression through analysis of RNA-seq data of 667 glioma patients. In the Moroccan cohort, VISTA gene expression was significantly upregulated in glioma tissues related to PBMC of healthy donors. This high expression was specific to patient tissues since VISTA expression in PBMC was low when assessed either before or after surgery. Besides, VISTA exhibited higher expression levels in grade III/IV relative to grade I/II glioma patients. Interestingly, VISTA correlated positively with PD-1 expression. PD-1 also showed elevated expressions in higher glioma grades. The TCGA cohort corroborated these observations. Indeed, VISTA was also found to be strongly expressed in high grades. It was positively correlated with other critical immune checkpoints. Finally, increased VISTA transcript levels were associated with weak overall survival of glioma patients. Our study highlighted a correlation between high levels of VISTA expression and poor prognosis in glioma patients. VISTA might be involved in glioma progression and could be considered as a possible new therapeutic target, especially in advanced gliomas.

Figure 1

VISTA gene expression was upregulated in glioma tissues relative to PBMC of healthy donors (Moroccan cohort). VISTA and PD-1 transcripts expression were performed using RT-PCR analysis. (a) VISTA gene strongly expressed in grade III–IV compared with grade I–II of glioma patients. (b) VISTA mRNA expressionwas significantly increased in advanced glioma (grade IV) compared to grade II. (c) PD-1was highly expressed in advanced glioma grading (grade III–IV). (d) Elevated expression of VISTA gene in glioma tissues (G-Tissues) compared to PBMC of healthy donors (H-PBMC) and PBMC of glioma patients before (pre-G PBMC) and after surgery (post-G PBMC). (e) VISTA was upregulated in high grade glioma tissues (grade III–IV) relative to PBMC of the same patients. Paired t-test was used to make a statistical comparison of VISTA and PD-1 expression between groups. p value less than 0.05 was considered as statistically significant for all statistical analyses.

Figure 2

Immunohistochemical staining of VISTA revealed elevated expression levels in high grade gliomas. Representative staining intensity of VISTA protein was detected in human glioma tissues (Moroccan cohort) using immunohistochemistry assay. (a) Negative control staining in glioma case with Rabbit IgG Isotype Control (magnification × 20). (b) Negative staining of VISTA in low grade glioma (grade I). (c) Positive staining of VISTA in high grade glioma (Glioblastoma IV) (magnification × 20). (d) Positive staining of VISTA on endothelial cells (magnification × 20). (e) Positive staining of VISTA on glioma cells (magnification × 40). (f) Positive staining of VISTA on immune cells in a glioblastoma case (magnification × 20). (g) Expression of VISTA according to glioma grades. Statistical analysis was performed by using a t-test to compare the expression of VISTA between different grades of glioma patients. p value less than 0.05 was considered as statistically significant for all statistical analyses.

Figure 3

VISTA transcripts strongly expressed in high glioma grades in the TCGA cohort and positively correlated with critical immune checkpoint regulators. RNAseq of 667 glioma patients of different grades were analyzed using TCGA dataset. (a) VISTA mRNA evaluation revealed high expression in advanced gliomas (grade IV). (b) Astrocytomas showed elevated expression of VISTA compared to oligoastrocytomas and oligodendrogliomas. (c) Mesenchymal and Neural glioma subtypes presented high VISTA expression in comparison with classical and proneural. (d) PD-1, Tim-3 and LAG-3 were upregulated in high grade glioma (glioblastoma).T-test was applied to compare gene expression between different grades and groups of glioma patients. p value less than 0.05 was considered as statistically significant for all statistical analyses.

Figure 4

VISTA expression positively correlated with critical immune checkpoint regulators. (a) VISTA expression was positively correlated with PD-1, Tim-3 and LAG-3. (b) VISTA gene exhibited the highest expression in comparison with other immune checkpoints (Tim-3, PD-1, CTLA-4, LAG-3, TIGIT). Spearman correlation test was used to examine the association of relative gene expression levels between VISTA and other immune checkpoints. Also, the one-way ANOVA test was used to determine the statistical significance of gene expression among different groups of glioma patients. p value less than 0.05 was considered as statistically significant for all statistical analyses.

Figure 5

Increased VISTA transcripts level associated to a poor prognosis of glioma patients in the TCGA dataset. A binary clustering of patients has been performed, using the median as a cut off for patient stratification: one group with a high expression of VISTA and a second one with lower expression. (a) CD4 and CD8 mRNA expression were both elevated in high versus low VISTA expression. (b) The immunomodulatory genes (TGF-β, IL-10 and IFNγ) showed high expression in glioma patients with higher levels of VISTA gene expression. (c) High VISTA expression levels associated with a bad overall survival. (d) Elevated expression of both VISTA and PD-1 correlated with a weak survival. Paired t-test was used to make statistical comparison of gene expression between groups. The prognostic value of VISTA gene was investigated by Log-rank test using the Kaplan–Meier plot. p value less than 0.05 was considered as statistically significant for all statistical analyses.