| ObjectiveGlioblastoma (GBM) is one of the most prevalent tumors in central nervous system. Probably due to tumor immune escape, the survival is still very poor under standard treatments. Regulatory T cells (Tregs), which usually lead to immunosuppression, have been found in glioma tissue. Some studies have focused on the prognostic value of Tregs in patients with glioblastoma but their results were controversial. Moreover, the differentiation mechanism of Tregs has been rarely reported. So in this article, we first tried to clarify the distribution of Tregs in glioblastoma, then analyzed their prognostic value, and investigated the potential differentiation mechanism from naive T cells to Tregs at last.Materials and methodsIn the first part, Western Blot was performed to detect the level of Foxp3 protein in brain trauma tissue, low grade glioma (LGG) and high grade glioma (HGG). Then Foxp3, CD8, p53, MGMT and Ki-67 were stained by immunohistochemistry on paraffin embedded tumor samples from 62 patients with glioblastoma. Following microscopic evaluation, the linear correlation between Foxp3 and CD8 was analyzed. Based on clinicopathologic data of the patients, the expression of Foxp3 was also compared among various subgroups.In the second part, we dichotomized the patients into two groups using the median density of Foxp3, CD8 or their ratio as cutoff value and performed survival analysis with Kaplan-Meier method. Moreover, their prognostic value was also tested in subgroups classified according to p53, MGMT and Ki-67. At last, all clinicopathologic and immune factors were included in univariate and multivariate analysis.In the third part, FlowCytomix and ELISA were performed on serum, cerebrospinal fluid and homogenate of patients with glioma to compare the concentrations of IL-6 and IL-10. Then using different levels of IL-6 or IL-10 in culture medium of naive CD4+T cells for 72 hours, flowcytometry was conducted to detect the percentage of CD25+Foxp3+ cells. After separated from fresh GBM tissue, glioma stem cells (GSCs), CD133-cells and their supernatant were used to incubate naive CD4+T cells. Then the percentage of Tregs was detected as above. Moreover, we also investigated the effect of B7-H4 positive macrophages on Tregs.ResultsHigher expression of Foxp3 protein were found in high grade glioma with little in brain trauma tissue. Foxp3 was mainly stained on nucleus with lower density than CD8 (8.6 vs 49.6 per mm2, P<0.001). And the two parameters were significantly correlated (rs=0.375、P=0.003). In addition, no relationship was found between Foxp3 density and clinicopathologic factors.The median PFS was 9 months in patients with higher Foxp3 expression and 12 months in patients with lower Foxp3 expression. The difference between the two groups was significant (P=0.001) and OS analysis showed similar results (16 vs 20 months, P=0.003). Comparing the two groups categorized by CD8 expression, significant difference was reached for neither PFS nor OS. The median PFS of patients with higher Foxp3+/CD8+was obviously shorter than that of patients with lower Foxp3+/CD8+(9 vs 12 months, P=0.031). The prognostic value of Foxp3 was various under different pathologic subgroups. For example, the correlation between Foxp3 and survival was only valid in Ki-67 negative group, other than the positive group. Later, multivariate analysis showed that Foxp3 density was an independent prognostic factor for both PFS and OS.IL-6 concentration was not found to be significantly different between the body fluid of LGG and HGG patients, but obviously elevated in the tumor homogenate of HGG patients. The percentage of Treg in CD4+cells was raised significantly after the stimulation of IL-6 at 1 ng/ml (P<0.05), and in a greater degree when 5 ng/ml was established (P<0.01). However, continuing increase of IL-6 did not upgrade Treg any more. IL-10 concentration was also not significantly different between the body fluid of LGG and HGG patients. It was elevated in the tumor homogenate of HGG patients, but not reaching statistical significance. And various levels of IL-10 did not affect the percentage of Tregs. Compared with CD133-glioma cells and their supernatant, GSCs and supernatant obviously induced the differentiation from naive CD4+ cells to Tregs, but no difference was found between these two groups. Moreover, B7-H4 positive macrophages could also upgrade the level of Tregs while the negative ones could not.ConclusionsThere exist more Tregs in HGG tumor tissue than the LGG one, and the Treg density is positively correlated with that of CD8+cells. As an independent prognostic factor for GBM patients, Treg infiltration usually predicts poor outcome. In glioma microenvironment, IL-6, GSCs and B7-H4 positive macrophages can all promote the differentiation from naive CD4+cells to Tregs. |
PDF Full Text Request