Treg depletion with a low-dose metronomic temozolomide regimen in a rat glioma model

Abstract

Background: CD4+CD25+ regulatory T cells (Treg), which constitute about 2-3% of CD4+ human T cells, are the main contributors to the maintenance of immune tolerance. Cancer patients, including glioblastoma patients, bear increased number of circulating and tumor infiltrating Treg that exert functional inhibition on tumor-specific T cells. Temozolomide (TMZ) is one of the most effective chemotherapeutic agents in glioblastoma (GBM). Lymphopenia is a common side effect of TMZ treatment, but to what extent the Treg compartment is affected by this chemotherapy has been poorly investigated. We therefore studied the impact of various TMZ regimens on Treg cell population in a TMZ-resistant rat model of glioma.

Methods: RG2 glioma cells were implanted s.c. in Fischer rats. Twelve days after tumor implantation, TMZ was administered orally with schedules designed to mimic the TMZ regimens currently used in humans: 30 mg/kg per day for 5 days, or 10 mg/kg per day for 21 days. In addition, two metronomic regimens with low-dose TMZ (2 and 0.5 mg/kg per day for 21 days) were evaluated. Splenocytes and tumor infiltrating lymphocytes were analysed by flow cytometry using CD3, CD4, CD25, and Foxp3 mAbs. Statistical significance was determined by the Mann-Whitney U test, the Student's t test or the ANOVA test.

Results: In the spleen of tumor-bearing animals, low-dose TMZ metronomic regimens (0.5 and 2 mg/kg for 21 days) induced a significant decrease of Treg/CD4+ ratios (13 +/- 2; p < 0.01, 14 +/- 3; p < 0.05, respectively, vs. 19 +/- 5 for controls). On the contrary, high-dose TMZ regimen (10 mg/kg per day for 21 days or 30 mg/kg for 5 days) did not significantly modify the percentage of Treg/CD4+. Within tumors, treatment with the 0.5 mg/kg TMZ regimen induced a slight and nearly significant decrease in the percentage of Treg/CD4+ after a 2 to 3-week treatment (24 +/- 9 vs. 35 +/- 11; p = 0.06). Treg depletion induced by the low-dose metronomic TMZ regimen was accompanied by a decreased suppressive function of the remaining Treg cells as assessed by an in vitro functional test. Treatment with 0.5 mg/kg metronomic TMZ reduced tumor progression when compared to untreated animals but the effect did not reach statistical significance, indicating that Treg depletion alone is not sufficient to significantly impact tumor growth in our model of fully established tumor.

Conclusions: A low-dose metronomic TMZ regimen, but not a standard TMZ regimen, reduced the number of circulating Tregs. These results can have clinical applications for immunotherapeutic approaches in GBM.

Fig. 1

Comparison of the Treg/CD4+ levels in the spleen of tumor-free rats (open circles, n = 9) and tumor-bearing rats (filled circles, n = 8). FACScan analysis of splenocytes was performed 32 days after RG2 tumor implantation. Treg/CD4+ levels were found to be higher in tumor-bearing animals (19 ± 5) than in tumor-free animals (14 ± 4), (p = 0.07)

Fig. 2

Comparison of the Treg/CD4+ levels (mean ± SD) in the spleen of RG2 tumor-bearing rats (7–9 rats per group) treated either by a 5 days/week regimen of TMZ (0.5, 2 or 10 mg/kg per day) for 3 weeks or by a 5-day standard regimen of TMZ (30 mg/kg per day). Tumors were implanted on day-12, 5 days/week regimens were initiated on day 0, and standard regimen of TMZ was initiated on day 14. Animals were sacrificed 72 h after receiving the last dose of treatment. (* p < 0.05; ** p < 0.01)

Fig. 3

Comparative study of the characterization of Treg splenocytes by CD25 surface staining or by Foxp3 intracellular staining. Four groups of three rats were either inoculated with RG2 cells on day-12 or left tumor-free (Naive). Treatment, which consisted of vehicle alone (Untreated), TMZ 30 mg/kg per day for 5 days (Std TMZ) or TMZ 0.5 mg/kg per day for 3 weeks (Metronomic TMZ) was administered to tumor-bearing rats. Animals were sacrificed on day 21. Isolated splenocytes were stained with antiCD3, antiCD4, and antiCD25 or with antiCD3, antiCD4, and antiFoxp3 mAbs and then submitted to flow cytometry analysis. a Results expressed as mean ± SD of the Treg/CD4+ levels (%) in the four groups of rats, Treg being either characterized as CD4+CD25+ cells (open bars) or as CD4+Foxp3+ cells (filled bars). b FCM analysis of spleen cells isolated from an untreated (left) and a metronomic TMZ-treated rat (right), both bearing RG2 tumors. Cells were surface stained with antiCD3-FITC and antiCD4-APC, then submitted to intracellular staining with antiFoxp3-PE. These plots (gated on the CD3+ population) are representative of the results obtained with three rats in each group (percentage of cells in the CD4+ quadrants are indicated)

Fig. 4

Comparison of the impact of standard and metronomic TMZ regimens on Treg function. Four groups of three rats were either inoculated with RG2 cells on day-12 or left tumor-free (Naive). Treatment, which consisted of vehicle alone (Untreated), TMZ 30 mg/kg per day for 5 days (Std TMZ) or TMZ 0.5 mg/kg per day for 3 weeks (Metronomic TMZ) was administered to tumor-bearing rats. Animals were sacrificed on day 21 and T cells were isolated from the collected spleens. 2 x 10⁶ splenic T cells depleted (open bars) or not (filled bars) of Treg were incubated for 3 days with antiCD3 mAb. Supernatants of these cultures were harvested and IFN-γ concentrations were determined by an ELISA test. Results are expressed as mean ± SD of IFN-γ levels measured in three splenic T cell cultures obtained from three different rats. IFN-γ levels observed in cultures depleted or not of Treg were compared within each group. NS not significant; * p < 0.05; paired Student’s t test

Fig. 5

Effect of a low-dose metronomic TMZ regimen on tumor growth. Rats were inoculated with RG2 cells on day-12. Treatment, which consisted of vehicle alone (filled circles) or TMZ 0.5 mg/kg per day (open squares) was initiated on day 0. Tumor growth was assessed twice a week by size measurement with a calliper, and tumor volume was estimated using the standard formula: p/6 × length × width². Results are expressed as mean ± SEM of tumor volumes measured in 30 rats per group, by compilation of several experiments