Increased proportion of FoxP3+ regulatory T cells in tumor infiltrating lymphocytes is associated with tumor recurrence and reduced survival in patients with glioblastoma

Abstract

Glioblastoma multiforme (GBM) is an aggressive malignancy associated with profound host immunosuppression mediated in part by FoxP3 expressing regulatory CD4+ T lymphocytes (Tregs) that down-regulate anti-tumor immunity. In order to assess whether FoxP3 was an independent driver differentially expressed in primary versus recurrent GBMs, we stained resected primary and recurrent GBM tumors for CD3, CD4, CD8 and FoxP3 expression using standard immunohistochemistry. Slides were scanned with a high-resolution scanner (ScanScope CS; Aperio), and image analysis software (Aperio ScanScope) was used to enumerate lymphocyte subpopulations allowing for high-throughput analysis and bypassing manual selection bias. As shown in previous studies, enumeration of individual lymphocyte populations did not correlate with clinical outcomes in patients with GBM. However, the CD4+ to regulatory FoxP3+ T cell ratio was diminished in recurrent disease, and increased CD3 and CD8+ to regulatory T cell ratios showed a positive correlation with survival outcomes in primary GBM. These results suggest that while absolute numbers of tumor infiltrating lymphocytes may not be informative for predicting clinical outcomes in patients with GBM, the effective balance of CD3, CD4 and CD8+ T cells to immunosuppressive FoxP3+ regulatory cells may influence clinical outcomes in this patient population.

Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest with the research presented in this paper.

Figures

Fig. 1

Aperio Analysis of GBM specimens stained for T cell markers by IHC. Primary GBM pathology specimen was stained for FoxP3 using standard IHC (a) and analyzed by Aperio (b) with images superimposed (c). FoxP3 expressing cells by IHC are quantified as strong positive pixels shown in red, while background is shown as blue (b). CD3, CD4, CD8 or FoxP3 expressing cells were manually counted demonstrating strong positive concordance with Aperio quantification of IHC (d)

Fig. 2

Absolute cell counts and cell count ratios in primary versus recurrent GBM. Primary and recurrent GBM pathology specimens were stained for CD3, CD4, CD8 and FoxP3+ markers and quantified using Aperio software. Absolute counts were divided by surface area of each specimen to standardize measurements, and the ratio of CD4, CD8 and FoxP3+ cells was measured over CD3+ cells. Absolute counts alone were not different between primary and recurrent GBM (a, b, c), but when expressed as percentage of T cell subsets over total CD3+ counts, recurrent GBMs had decreased proportions of CD4+/CD3+ cells (d); however, CD8+ and FoxP3+ cells remained unchanged when expressed as a percentage over total CD3+ cells (e, f)

Fig. 3

CD3, CD4 and CD8+ to FoxP3+ ratio in primary versus recurrent GBM. Primary and recurrent GBM pathology specimens were stained for CD3, CD4, CD8 and FoxP3 markers and quantified using Aperio software. Ratios of CD3, CD4 and CD8+ cells were measured over FoxP3+ cells. There is a higher ratio of CD4/FoxP3+ cells in primary versus recurrent GBMs (b) but no difference when comparing CD3/FoxP3+ and CD8/FoxP3+ ratios (a and c)

Fig. 4

Correlation of CD3, CD4 and CD8+ over FoxP3+ T cell ratios and survival in primary GBM. Primary GBM pathology specimens were stained for CD3, CD4, CD8 and FoxP3 markers and quantified using Aperio software. Survival (in days) of patients was plotted versus ratios of CD3, CD4 and CD8+ cells over FoxP3+ cells. Patients still alive at time of data analysis are represented by a clear circle. There is a positive correlation between survival and increased CD3+ to FoxP3+ ratios (a; R value = 0.7091) and CD8+ to Foxp3+ ratios (c; R value = 0.6343) but no correlation between survival and CD4+ to FoxP3+ ratios (b; R value = 0.3450)