Selective depletion of regulatory T cell subsets by docetaxel treatment in patients with nonsmall cell lung cancer

Abstract

Regulatory T (Treg) cells are potent suppressors that maintain immune homeostasis. Accumulation of Treg can inhibit effective immune responses in cancer patients, leading to tumor development and progression. Despite direct cytotoxicity, several chemotherapeutic drugs have been reported to deplete Treg cells for better prognosis for cancer patients. Treg cells are a heterogenous population with at least three different subsets, nonsuppressive, resting, and activated Treg cells. However, the characteristics of Treg cell subsets in lung cancer patients and how chemotherapy affects Treg cells remain elusive. In this study, we first analyzed Treg cell subsets in peripheral blood samples from 40 nonsmall cell lung cancer (NSCLC) patients and 20 healthy donors. Treg cells, specifically activated Treg cell subset, significantly increased in patients with NSCLC. Compared to nonsuppressive Treg cells, activated Treg cells expressed higher level of CD39 and predominantly produced inhibitory cytokines. In vitro assay showed that docetaxel reduced all three subsets of Treg cells. More importantly, we found docetaxel-based chemotherapy significantly decreased all three Treg subsets after 4 cycles of treatment in 17 NSCLC patients. Taken together, this study revealed dynamic changes of various Treg cell subsets in NSCLC patients before and after chemotherapy, providing activated Treg cells as a potential target for chemotherapy.

Figure 1

aTreg but not rTreg and non-Treg cells increased in NSCLC patients. (a) CD4⁺Foxp3⁺ T cells and three subsets of Treg cells from PBMCs were isolated and analyzed by FACS. (b) The percentages of Treg cells (CD4⁺Foxp3⁺ T (total Treg) cells, CD4⁺CD45RA⁻Foxp3^hi (aTreg) cells, CD4⁺CD45RA⁻Foxp3^lo (non-Treg) cells, and CD4⁺CD45RA⁺Foxp3^lo (rTreg) cells) in CD4⁺ T cells were calculated after FACS analysis. HD: healthy donor, n = 20; NSCLC: nonsmall cell lung cancer, n = 40. Each dot represents one individual sample. **P < 0.01 and ***P < 0.001 for statistical analysis by Student's t-test.

Figure 2

aTreg cells expressed higher immunosuppressive marker CD39 in NSCLC patients and secreted suppressive cytokines. (a) PBMCs were collected from NSCLC patients. The phenotype marker of CD39 was evaluated in the three subsets of CD4⁺Foxp3⁺ Treg cells, including aTreg, non-Treg, and rTreg cells. The dot plots (left) represent the expression of CD39 in each group. The bar figures (right) represent the mean percentage of each population ± standard error of mean. (b) These cells were also stimulated in vitro and the cytokine profiles including TGF-β and IFN-γ were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001 by paired t-test.

Figure 3

aTreg cells had a higher level in patients with advanced NSCLC. NSCLC patients were grouped according to clinical stage and pathology. (a) The percentages of CD4⁺Foxp3⁺ T cells, aTreg cells, non-Treg cells, and rTreg cells were compared in PBMCs of NSCLC patients at stages I-II and III-IV. (b) The 4 groups of Treg cells were compared in PBMCs of NSCLC patients between adenocarcinoma and squamous carcinoma. Statistical analysis was determined by one-way ANOVA. *P < 0.05.

Figure 4

The three Treg subsets reduced after being treated with docetaxel in vitro. (a) PBMCs from NSCLC patients were staining with CD25 and Foxp3 antibody and analyzed by flow cytometry. (b) The peripheral blood of NSCLC patients was collected and CD4⁺CD25⁺ T cells were isolated by flow cytometric sorting. Three Treg subsets were defined with CD4⁺CD45RA⁻CD25^hi (aTreg) cells, CD4⁺CD45RA⁻CD25^lo (non-Treg) cells, and CD4⁺CD45RA⁺CD25^lo (rTreg) cells. The differences of three subsets with or without docetaxel were analyzed. *P < 0.05; **P < 0.01 by paired t-test.

Figure 5

Treg cell subsets of NSCLC patients were decreased after chemotherapy. The patients with NSCLC received 4 cycles of chemotherapy and the peripheral blood was collected 1 day before the first cycle and 2 weeks after each cycle. Three subsets were analyzed by FACS. Statistical analysis was determined by randomized block design ANOVA. *P < 0.05.