An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme - PubMed (original) (raw)

Comparative Study

An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme

Abdeljabar El Andaloussi et al. Neuro Oncol. 2006 Jul.

Abstract

The subpopulation of CD4+CD25+ immunoregulatory T (Tr) cells constitutes 5%-10% of CD4+ cells in humans. These cells play a crucial role in the control of tumor immune response. In this study, we evaluated the distribution of Tr cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme and examined the difference between the brain and autologous blood with respect to Tr cells. Glioma samples from 10 patients were classified as WHO grade IV astrocytoma. Control samples were obtained from patients undergoing resection of a seizure focus. The samples were analyzed by flow cytometry to determine the frequency of Tr cells and by real-time PCR for forkhead box P3 (FOXP3) expression. We then examined the expression of CD62L, CD45RO, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and assessed the functionality of Tr cells in vitro. There was a significant difference in the number of FOXP3-expressing CD4+CD25+ T cells within glioma-infiltrating lymphocytes as compared to controls (P < 0.01). This difference was further observed in studies of autologous patient blood and control blood. The expression level of FOXP3 mRNA was high in Tr cells and weak in CD4+CD25-T cells. Moreover, the expression of CD62L and CTLA-4 was elevated in glioma Tr cells as compared to that in the controls. These cells were also CD45RO positive. Functional assays confirmed the suppressive activity of Tr cells in patients with glioma. The expression of CD4+CD25+FOXP3+ T cells was significantly higher in patients with glioblastoma multiforme than in controls. This increase in the frequency of Tr cells that display suppressive activity might play a role in modulation of the immune response against glioma. In light of these findings, Tr cells may represent a potential target for immunotherapy of malignant brain tumors.

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Figures

Fig. 1

T-cell infiltrate in brain and blood of patients with malignant glioma. As compared to healthy brain (A), there is an increased percentage of tumor-infiltrating lymphocytes in brain with glioma (B). The frequency of TILs was significantly increased (P < 0.01) in brain tumors (mean, 17.7% ± 2.7%; range, 12%–21.4%) versus control brain (mean, 0.5% ± 0.28%; range, 0–0.85%). In addition, in comparison with percentage of total lymphocytes in the blood of control patients (C) (mean, 10.3% ± 1.51%; range, 7.8%–12.4%), there is an increased percentage of total lymphocytes in the blood of glioma patients (D) (mean, 16.8% ± 1.9%; range, 13.6%–20%) (P < 0.01). Cells were gated on lymphocytes via their forward and side scatter properties.

Fig. 2

Regulatory T cell frequency in total CD3+ T cells of brain. Flow cytometric analysis of CD3+ T cells (I) and CD4+CD25+ T cells (II) from brain with malignant glioma (A) and control brain (B). The mean of Tr cells in glioma-infiltrating lymphocytes was 24.7% ± 2.54% (range, 19.1%–27%), and Tr cells were absent from control brain. Representative FACS plots are shown demonstrating (I) CD3 and (II) CD4 versus CD25 staining.

Fig. 3

Analysis of CD4+CD25+ T cells in peripheral blood. Autologous blood from glioma patient (A) versus control blood (B). Representative FACS plots are shown demonstrating CD3 (I) and CD4 and CD25 (II) staining. The frequency of Tr cells in the glioma patient’s blood was increased when compared with the frequency observed in control blood (8.56% ± 2.46% [range, 6.4%–12.4%] vs. 0.48 ± 0.13% [range, 0.26%–0.7%]; P < 0.05). Cumulative results of flow cytometric analyses of regulatory T cells are shown as the percentage of total CD4+ or CD3+ T cells.

Fig. 4

Expression of intracellular FOXP3 in total CD4+ cells. Higher levels of FOXP3 expression in CD4+CD25+ cells were observed in (B) regulatory T cells isolated from tumor tissue (55.1% ± 1.88%; range, 51%–62.4%) versus (A) autologous patient blood (33.4% ± 1.95%; range, 29.7%–38.4%) versus (C) control blood (15.6% ± 0.76%; range, 13.2%–17.1%) (P < 0.01). (D) Comparison of FOXP3 and CD25 expression on Tr in TILs, autologous blood, and peripheral blood of control. Abbreviations: HB, healthy blood; AB, autologous blood; NB, control brain; GB, glioma brain.

Fig. 5

Level of FOXP3 expression in CD4+ T cell subsets. FOXP3 quantification by real-time PCR (A) and semiquantitative PCR (B) in CD4+ T cells. The real-time PCR data shows that the level of FOXP3 mRNA was fivefold to sixfold higher in CD4+CD25+ cells (A). This difference was significant with respect to control (P < 0.05). Lines 1 and 2 consisted of CD4+CD25− and CD4+CD25+ sorted from autologous blood, and lines 3 and 4 consisted of CD4+CD25− and CD4+CD25+ sorted from control blood donor. CD4+CD25− and CD4+CD25+ were sorted separately from PBMCs with more than 97% purity.

Fig. 6

Regulatory T cells show suppressor phenotype. The expression of CD62L after gating of CD4+CD25+ or CD4+CD25− cells in autologous blood of patients (A) and control blood (B). The left panels of rows A and B show, respectively, an expansion of CD25−CD62L− subsets in glioma patient (mean, 75.2% ± 4.03%; range, 70%–81%) compared to control blood (mean, 63.6% ± 3.74%; range, 58%–69%) (P < 0.01). The same expansion of CD25+CD62L+ was seen in glioma patients (mean, 90.3% ± 2.83%; range, 88%–97%) versus controls (mean, 83.5% ± 4.34%; range, 77%–88%) (P < 0.01). The cells were stained with three-color fluorescence and analyzed by fluorescent flow cytometry.

Fig. 7

Expression of CD45RO and CTLA-4 on CD4+ T cells. Flow cytometric analysis of CD45RO and CTLA-4 (CD152) on CD4+CD25+ T cells. A and C. Control blood. B and D. Autologous patient blood. In autologous blood of glioma patients, the pool of memory CD4+CD45RO+ T cells was CD45RO positive (mean, 41.5% ± 1.73%; range, 38.5%–43%), but the expression level of CD45RO was less than in control donors (mean, 54% ± 2.45%; range, 50%–57%) (P < 0.02) (A and B). The CTLA-4 expression on CD4+CD25+ T cells in autologous blood and control blood was 76.8% ± 2.1% [range, 74%–80.5%] versus 50% ± 1.8% [range, 47.7%–52%], respectively (C and D) (P < 0.01).

Fig. 8

In vitro suppression assay. CD4+CD25− T cells were cocultured alone or with CD4+CD25+ T cells at different ratios and stimulated with anti-CD3 and anti-CD28. Proliferation was assessed by [3H]thymidine incorporation. The results represent the average [3H]thymidine incorporation (cpm) from five replicate wells per culture. A. Gates used for sorting Tr cells. B. Suppressor activity of CD4+CD25+ T cells.

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An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme - PubMed (original) (raw)