Effects of nilotinib on regulatory T cells: the dose matters

Abstract

Background: Nilotinib is a tyrosine kinase inhibitor with high target specificity. Here, we characterized the effects of nilotinib for the first time on CD4+CD25+ regulatory T cells (Tregs) which regulate anti-tumor/leukemia immune responses.

Design and methods: Carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2-deoxy -uridine (BrdU) were used to assess the proliferation and cell cycle distribution of Tregs. The expression of the transcription factor forkhead box P3 (FoxP3) and the glucocorticoid-induced tumor necrosis factor receptor (GITR) were measured by flow cytometry. Western blotting analysis was used to detect the effects of nilotinib on the signal transduction cascade of T-cell receptor (TCR) in Tregs.

Results: Nilotinib inhibited the proliferation and suppressive capacity of Tregs in a dose-dependent manner. However, the production of cytokines secreted by Tregs and CD4+CD25- T cells was only inhibited at high concentrations of nilotinib exceeding the mean therapeutic serum concentrations of the drug in patients. Only high doses of nilotinib arrested both Tregs and CD4+CD25- T cells in the G0/G1 phase and down-regulated the expression of FoxP3 and GITR. In western blotting analysis, nilotinib did not show significant inhibitory effects on TCR signaling events in Tregs and CD4+CD25- T cells.

Conclusions: These findings indicate that nilotinib does not hamper the function of Tregs at clinical relevant doses, while long-term administration of nilotinib still needs to be investigated.

Figure 1

Nilotinib inhibits the proliferation of CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells in a dose-dependent manner. CD4⁺CD25⁺ T cells or CD4+CD25^- T cells were labeled with CFSE and cultured with different concentrations of nilotinib as indicated. After 4 days incubation, cells were harvested and stained with CD25 and FoxP3. Cell division was assessed by the dilution of CFSE. Panels A and B show the combined results from four independent experiments with similar results. Displayed are mean values ± SD. Panels C and D display the results of a representative experiment.

Figure 2

Nilotinib does not hamper immunosuppressive capacity of CD4⁺CD25⁺ T cells at clinical relevant doses. Panel A: Purified CD4⁺CD25⁺ T cells or CD4⁺CD25^- T cells (Teff) were labeled with CFSE. CFSE⁺-labeled CD4⁺CD25^- T cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of nilotinib. The proliferation of CD4⁺CD25^- T cells was measured by the division of CFSE. Combined results from three independent experiments were shown. Panel B: CD4⁺CD25⁺ T cells were first incubated with different concentrations of nilotinib overnight in the presence of IL-2. After washing cells for three times, CD4⁺CD25⁺ T cells were co-cultured with CFSE⁺-labeled CD4⁺CD25^- T cells as a ratio of 1:1. Cell division was assessed by levels of CFSE dilution.

Figure 3

Nilotinib does not induce apoptosis on CD4⁺CD25⁺ T cells and CD4⁺CD25^-T cells. CD4⁺CD25⁺ T cells (Panel A) and CD4⁺CD25^-T cells (Panel B) were stimulated with anti-CD3, anti-CD28 and IL-2 with different concentrations of nilotinib as indicated for 48 h. Cells were then harvested and stained with Annexin V* FITC and PI. Apoptotic cells were defined by flow cytometry as Annexin V positive and PI negative cells.

Figure 4

Nilotinib inhibits the cytokine production by CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells. Panel A: CD4⁺CD25⁺ T cells were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of 25 μM nilotinib. After 4 days incubation, supernatants were collected for cytokine analysis. The high intensity spots in the three corners are positive controls, the lower right contains the negative controls. Panel B: Graphs of the relative density (subtracted the average background signals) are shown for selected cytokines. Array images are shown in panel A and profiles created by quantifying the background-subtracted mean spot pixel densities are identified as shown in Panel B by using image analysis software. Panel C + D: The cytokine production of CD4⁺CD25^- T cells treated with nilotinib was analyzed by above methods.

Figure 5

High concentrations of nilotinib down-regulate the expression of FoxP3 and GITR by CD4⁺CD25⁺ T cells. Purified CD4⁺CD25⁺ T cells were unstimulated or stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of nilotinib as indicated. Then cells were stained with FoxP3*PE or GITR*PE according to the manufacturer's instruction for fixation, permeabilization, and staining, after cells were stained for surface expression of CD4 and CD25. Nilotinib significantly decreased the intracellular expression of FoxP3 and GITR on CD4⁺CD25⁺ T cells in a dose-dependent manner at concentrations of 25 μM. Data are representative of four independent experiments with similar results. Displayed are mean values ± SD.

Figure 6

Nilotinib does not inhibit the signaling events on TCR in CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells. Panel A: Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were incubated with nilotinib for 1 hour. Then cells were not stimulated or stimulated with anti-CD3/CD28 for 15 minutes. Whole cell lysates were analyzed by western blotting for the phosphorylation levels of Lck and Zap70. (1. non stimulated CD4⁺CD25⁺ T cells; 2. non stimulated CD4⁺CD25^- T cells; 3. stimulated CD4⁺CD25⁺ T cells; 4. stimulated CD4⁺CD25^- T cells; 5. stimulated CD4⁺CD25⁺ T cells plus nilotinib; 6. stimulated CD4⁺CD25^- T cells plus nilotinib; 7. stimulated CD4⁺CD25^- T cells plus nilotinib; 8. stimulated CD4⁺CD25^- T cells plus nilotinib; the respective concentration of nilotinib is indicated in the figure.) Panel B: Comparison of imatinib, nilotinib and dasatinib on the effects of TCR, Src and NF-κB signaling molecules in the Jurkat cell line. Compared with imatinib and dasatinib, nilotinib did not show obvious inhibitory effects in Jurkat T cells, while dasatinib showed the highest inhibitory potency among the three drugs. Data are representative of three experiments with similar results.