Immune modulation of CD4+CD25+ regulatory T cells by zoledronic acid

Abstract

Background: CD4⁺CD25⁺ regulatory T (Treg) cells suppress tumor immunity by inhibiting immune cells. Manipulation of Treg cells represents a new strategy for cancer treatment. Zoledronic acid (ZA), a nitrogen-containing bisphosphonate, inhibits the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) on osteoblasts to inhibit osteoclastogenesis. In a mouse model of bisphosphonate-related osteonecrosis of the jaw, administration of ZA suppressed Treg-cell activity and activated inflammatory Th17 cells. However, the interaction between ZA and Treg cells remained unclear. This study investigated the immune modulation of Treg cells by ZA.

Methods: Flow cytometry was used to analyze the phenotypic and immunosuppressive characteristics of Treg cells treated with ZA. Chemotactic migration was evaluated using transwell assays. Quantitative real-time PCR (qRT-PCR) was used to investigate the effect of ZA on the expression of suppressive molecules by Treg cells.

Results: Proliferation of isolated Treg cells in culture was inhibited by ZA, although ZA did not induce apoptosis. qRT-PCR and flow cytometry showed that ZA significantly downregulated the expression of CCR4, CTLA4, PD-1 and RANKL on Treg cells. Chemotactic migration and immunosuppressive functions were also significantly attenuated in Treg cells pretreated with ZA, and these effects were dose-dependent. Co-culture with Treg cells significantly increased the migration rate of breast cancer cells, while pretreatment of Treg cells with ZA attenuated this effect.

Conclusions: Our findings demonstrated that ZA acted as an immune modulator by significantly inhibiting the expansion, migration, immunosuppressive function and pro-metastatic ability of Treg cells. Immunomodulation of Treg cells by ZA represents a new strategy for cancer therapy.

Keywords: Breast cancer; Immunomodulation; Regulatory T cells; Zoledronic acid.

Fig. 1

ZA inhibits Treg cells proliferation and induces cell injury. a Expanded Treg cells were labeled with CFSE and cultured in Treg cell medium with or without 10 μM ZA. b Treg cell proliferation curves were measured based on the percentage of cells with decreased fluorescence as compared to non-proliferating cells (0.38% at day 1). Data represent the mean values ± SEM and results from three independent experiments are shown. Statistical significance (P < 0.01) is denoted by **. c The morphology of Treg cells was analyzed by microscopy in 100× oil immersion after ZA treatment for 24 h

Fig. 2

ZA inhibits Treg cells chemotactic migration. Treg cells (5 × 10 ⁴) were pretreated with 0, 50 or 100 μM ZA for 4 h, and placed in the upper chambers. Migration of Treg cells into the lower chambers containing DMEM with 2% FBS or CM from MDA-MB-231 cells after 2 h was analyzed. The chemotaxis index shown compares migration with the response of control cells to DMEM with 2% FBS. Values are means ± SEM of results from three independent experiments in duplicate. *P < 0.05, ** p < 0.01, ***p < 0.001

Fig. 3

ZA influences the expression of markers associated with Treg cells suppressive function. Representative flow cytometry results of (a) CCR4 and (b) CTLA4 on Treg cells treated with 100 μM ZA (dotted line) and untreated Treg cells (shaded histogram). The negative control (unstained Treg cells) is shown as an open histogram. Median fluorescence intensity (MFI) is plotted against the ZA concentration (μM); the results from four independent experiments are shown. Significant difference in MFI determined using the one-way ANOZA;*p < 0.05

Fig. 4

Influence of ZA on mRNA expression by Treg cells. mRNA levels were evaluated by qRT-PCR in Treg cells treated with 0, 50, 100 μM ZA for 6 h. The gene expression values were normalized to GAPDH expression. The relative mRNA expression of (a) CCR4, (b) Foxp3, (c) TGFβ and (d) PD-1 was calculated from Treg cells treated with ZA compared with untreated Treg cells. Values shown are means ± SEM of results from four independent experiments performed in duplicate. *p < 0.05

Fig. 5

Effects of ZA on the ability of Treg cells to enhance MDA-MB-231 cell migration. a Representative pictures of migration (wound closing) of MDA-MB-231 cells grown as a mono-culture or co-culture with Treg cells at 0 and 12 h after wounding (100× magnification). b Quantification of the migration distance as a percentage of the control (MDA-MB-231 cells only). c The relative RANKL mRNA expression was evaluated by qRT-PCR from Treg cells treated with 0, 50, or 100 μM ZA for 6 h. The results from three independent experiments are shown. *P < 0.05, **p < 0.01

Fig. 6

ZA inhibits the immunosuppressive function of Treg cells. a Treg cells were treated with 0, 50, or 100 μM ZA for 24 h, and co-cultured with autologous anti-CD3/CD28-stimulated PBMCs for 7 h. The percentage of CD69 expression on effector T cells was analyzed by flow cytometry. b Treg cells were pretreated with CM from MDA-MB-231 cells for 72 h, followed by 0, 50, 100 μM ZA treatment for 24 h. The Tregs were then co-cultured with autologous anti-CD3/CD28-stimulated PBMCs for 7 h. The percentage of CD69 expression on effector T cells was analyzed by flow cytometry. c and d The percent suppression of the Tregs on effector T-cell without (c) and with (d) pretreated by CM from MDA-MB-231 cells after ZA treatment. The percent suppression was calculated by the following formula: 100–[(% CD69-positive in presence of Tregs/% CD69-positive in absence of Tregs) × 100]. Values shown are means ± SEM of results from four independent experiments performed in duplicate. *P < 0.05