Abstract
Expansion of CD4+CD25+ regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. Thymic stromal lymphopoietin (TSLP) contributes to the generation of natural Tregs in thymus. Therefore, the purpose of this report was to investigate the role of TSLP in the increasing prevalence of Tregs in lung cancer microenvironment. The expression ratio of TSLP protein in tumor tissues was significantly increased compared with that in benign lesion and non-cancer lung tissue. The prevalence of Tregs in tumor microenvironment was correlated with the expression of TSLP in lung cancer. Dendritic cells (DCs) were induced from peripheral blood mononuclear cells (PBMCs) collected from lung cancer patients and left unstimulated (imDCs) or exposed to hTSLP (TSLP-DCs) or LPS (LPS-DCs). TSLP-DCs expressed intermediate levels of CD83 and high levels of CD86, CD11C, and HLA-DR, which showed a characteristic of less mature DCs. TSLP-DCs secreted low levels of IL-6, IL-12, IL-10, TNF-α and IFN-γ, and high levels of TGF-β and MDC. The percentage of Tregs in CD4+CD25− T cells cocultured with TSLP-DCs group was statistically higher than that of LPS-DCs and imDCs. Transwell assays showed that TSLP-DCs exhibited increased ability to attract the migration of CD4+CD25− Tregs, when compared with imDCs. These results indicated that TSLP proteins were expressed in lung tumor tissue and correlated with the prevalence of Tregs. TSLP-DCs could induce CD4+CD25− T cells to differentiate into CD4+CD25+foxp3+ T cells and the migration of CD4+CD25+ T cells.
Keywords: TSLP, Lung cancer, Regulatory T cells, Dendritic cells, Immune suppression
Introduction
Cancer creates an immunosuppressive microenvironment to escape immune surveillance. It is important to identify the mechanisms underlying the interactions between cancer cells and the immune system, in order to develop better immunotherapeutic strategies. It has been reported that CD4+CD25+ regulatory T cells (Tregs), which could suppress the activity of lymphocytes and help the tumor cells to escape the host immune system, were increased in the peripheral blood or tumor microenvironment in patients with breast cancer [1], non-small cell lung cancer [2], gastrointestinal malignancies [3], and head–neck carcinoma [4]. Except for the natural thymus-derived CD4+CD25+Foxp3+ regulatory T cells, Tregs could also be induced from CD25− naïve CD4+ T cells both in vivo and ex vivo. This conversion requires cytokines such as TGF-β as well as suboptimal TCR stimulation and is thus regulated by the costimulatory status of antigen-presenting cells, such as dendritic cells [5–7]. Despite these previous findings, the mechanisms of the development of Tregs in cancer patients remain unknown. Liu et al. reported that TSLP, which is expressed in the Hassall’s corpuscles in the human thymic medulla, contributes to the generation of natural Tregs by inducing the differentiation of CD4+CD8−CD25− T cells into CD4+CD25+Foxp3+ Tregs [8]. We thereby speculated that TSLP might participate in the induction of Tregs in cancer patients. To test this hypothesis, we analyzed the expression of TSLP in tumor tissue and the correlation of TSLP expression with the number of Tregs in the tumor microenvironment in lung cancer patients. The effects of TSLP on the function of DCs and the induction of Tregs were also examined.
Materials and methods
Detection of TSLP protein by immunohistochemistry (IHC)
The expression of TSLP protein was detected in 46 cases of carcinoma, 20 cases of non-cancer lung tissue, and 15 cases of benign disease. The samples were used from a tissue bank of patients who had given written consent. Immunohistological staining was performed using the avidin–biotin–peroxidase method. Briefly, sections were deparaffinized by serial treatment. After blocking the endogenous peroxidase in 3% hydrogen peroxide and with 1% bovine serum albumin, the sections were incubated overnight at 4°C in the presence of sheep anti-human TSLP Ab (R&D Systems, USA) or control IgG. These sections were incubated with rabbit anti-sheep second antibody (Upstate USA). Distribution of peroxidase was revealed by incubating the sections in a solution containing 3% 3, 3-diaminobenzidine tetrahydrochloride before being counterstained with hematoxylin. Sections were evaluated in a blinded manner using light microscopy.
Detection of Foxp3+ Tregs by IHC
The staining of Foxp3 was carried out using mouse anti-human Foxp3 antibody (eBioscience, USA) and goat anti-mouse second antibody kit (Maixin, Fuzhou, China) as described above. The number of Foxp3+ Tregs was counted as follows: 10 fields containing positive cells from each section were counted for the number of positive cells per 100 cells in each field.
Induction of dendritic cells from PBMCs
Peripheral blood was collected from 10 lung cancer patients. PBMCs were isolated by centrifugation in Ficoll–Hypaque gradient. Ficoll-separated cells were incubated at 5 × 106 cells/ml in RPMI 1640 medium (Thermo Scientific HyClone, USA) supplemented with 10% FCS for 2 h before non-adherent cells were depleted. The non-adherent cells were collected and frozen at −80°C. The adherent cells were cultured in RPMI 1640 medium supplemented with 10% FCS (Thermo Scientific HyClone, USA), 1,000 U/ml GM-CSF (Peprotech, USA) and 1,000 U/ml IL-4 (Peprotech). DCs were subcultured every 3 days in GM-CSF and IL-4 containing medium. On day 6, DCs were left unstimulated or exposed 24 h to 15 ng/ml TSLP (R&D System, USA) or 1 μg/ml LPS (Sigma, USA).
Phenotype assays and cytokine quantification of DCs
After 24 h stimulation of DCs with human TSLP or LPS, the phenotype of TSLP-DCs, LPS-DCs, and imDCs was examined by flowcytometry assay; 5 × 105 dendritic cells were resuspended in 20 μl of 2% newborn calf serum and 1% sodium azide in phosphate-buffered saline (PBS) and incubated with 10 μl of antibodies against CD83-PE, CD86-PE, CD11c-PE, CD14-PE, and HLA-DR-FITC (BD Bioscience, USA) in separate tubes for 30 min at 4°C. After incubation, the cells were washed twice and resuspended in 1.0 ml of assay buffer. The fluorescence was analyzed by an Aria II flow cytometer (BD Bioscience, USA).
The supernatant of imDCs, TSLP-DCs, and LPS-DCs was collected, and the secreted levels of IL-6, IL-10, IL-12, TNF-α, IFN-γ, TGF-β (Sibozheng, Beijing, China), MDC and TARC (R&D system, USA) were measured by ELISA.
Phospho-specific protein microarray analysis
The activation status of JAK/STAT pathway in TSLP-DCs, LPS-DCs, and imDCs was analyzed using phospho-specific protein microarray (Full Moon Biosystems, Inc., CA, USA). Protein microarray analysis was carried out following the manufacturer’s protocol. Briefly, 50 μg of cell lysate in 60 μl of labeling buffer was labeled with 1.5μL biotin/DMF at room temperature for 2 h, before 25 μl stop reagent was added for 30 min incubation at room temperature. The antibody microarray was blocked with blocking solution at room temperature for 45 min and rinsed with Milli-Q grade water. The array was incubated with the biotin-labeled cell lysates for 1–2 h at room temperature and rinsed three times with wash solution for 10 min each. Finally, the biotin-labeled protein was detected using Cy3-streptavidin.
Western blotting analysis was performed to compare the early time course of activation of STAT proteins in TSLP-DCs, LPS-DCs, and imDCs. Dendritic cells were stimulated with TSLP or LPS for 15 min. The cells were collected and lysed with the Phosphosafe extraction reagent (EMD Chemicals, USA) for 5 min at room temperature. Cell lysates were centrifuged at 12,000 rpm for 5 min. The supernatant was collected, and the protein content of samples was determined with BCA protein Assay kit (Thermo Scitific, USA). Samples were subjected to Western blotting analysis with antibodies against STAT1, phosphorylated STAT1 (pSTAT1) (Tyr701), STAT3, pSTAT3 (Tyr705), STAT5, pSTAT5 (Tyrr694) (Cell Signaling Technology, USA), and β-actin (Santa Cruz, USA). Proteins (25 μg) were separated by a 10% SDS–PAGE gel using a Tris/glycine buffer. Following electrophoresis, proteins were transferred onto PVDF membrane and blocked by incubation in PBST (PBS, 0.01% Tween 20) containing BSA/milk overnight at 4°C. Membranes were incubated with primary and biotinylated antibodies at room temperature each for 2 h. Target proteins were detected with streptavidin–HRP and visualized with chemiluminescence.
Isolation of CD4+CD25− and CD4+CD25+ T cells from PBMCs
Isolation of CD4+CD25+ and CD4+CD25− T cells was performed in a two-step procedure (Miltenyi Biotec, Germany). Firstly, non-CD4+ cells were labeled with a cocktail of biotin-conjugated monoclonal antibodies against human CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ, and Glycophorin A. Then, the Anti-Biotin MicroBeads were added to the antibody-labeled cells, and the cells were subsequently depleted using a MACS column. In the second step, CD4+CD25+ T cells were directly labeled with CD25 MicroBeads and isolated by positive selection from the pre-enriched CD4+ T-cell fraction. The CD4+CD25− T cells were removed, and the CD4+CD25+ T cells were retained on the column and subsequently eluted for collection.
Effects of TSLP-DCs on inducing CD4+CD25− T cells differentiated into CD4+CD25+CD127− T cells
TSLP-DCs, LPS-DCs, and imDCs were cocultured with CD4+CD25− T cells at the ratio of 1:10 for 5 days. The percentage of CD4+CD25+CD127− T cells in different groups was examined by flow cytometry.
Effects of TSLP-DC on the migration of CD4+CD25+ T cells
The effects of TSLP-DCs, LPS-DCs, and imDCs on the migration of Tregs were analyzed by transwell assay (Corning, Netherlands). TSLP-DCs, LPS-DCs, or imDCs were plated at 5 × 105/well in 24-well dish. CD4+CD25+ T cells were seeded at 1 × 105/transwell in the top chamber of transwell insert with 3-μm pores. The staining of CD25-RPE in the lower chamber was analyzed, and positive cells were counted after 48 h.
Statistical analyses
All data were presented as means ± SD. The Student’s t test, ANOVA test, χ2 tests were used to determine the statistical significance. It was considered statistically significant when P < 0.05.
Results
Analysis of the expression of TSLP protein
The expression of TSLP protein was examined using immunohistochemistry (Fig. 1a). The positive rate in tumor tissue was 69.57%, which was significantly higher compared with 13.33% in benign lesion and 30.00% in non-cancer lung tissue (P < 0.05) (Fig. 1b). The correlations between the expression of TSLP protein and clinical characteristics were analyzed. The results indicated that the expression of TSLP protein was correlated with pathologic type, stage, tumor size, and lymph node (LN) metastasis (P < 0.05) (Table 1).
Fig. 1.
Expression of TSLP protein in lung cancer. a To analyze the expression of TSLP protein, 46 cases of tumor tissue, 15 cases of benign lesion, and 20 cases of non-cancer lung tissue were collected. The expression of TSLP protein in lung cancer and benign lesion was examined by immunohistochemistry. b The positive rate of TSLP in tumor tissue was 69.57%, which was significantly higher compared with that in benign lesion and non-cancer lung tissue
Table 1.
Correlation between the expression of TSLP protein in lung cancer and clinical characteristics
| Group | TSLP positive | TSLP negative | P |
|---|---|---|---|
| Pathology | |||
| Squamous carcinoma | 69.57% (16/23) | 30.43% (7/23) | 0.037 |
| Adenocarcinoma | 71.43% (10/14) | 28.57% (4/14) | |
| Adenosquamous carcinoma | 100.00% (6/6) | 0.00% (0/6) | |
| Small cell lung cancer (SCLC) | 0.00% (0/3) | 100.00% (3/3) | |
| Stage | |||
| I | 64.29% (9/14) | 35.71% (5/14) | 0.008 |
| II–IV | 71.87% (23/32) | 28.13% (9/32) | |
| Tumor size | |||
| ≤3 cm | 62.50% (5/8) | 37.50% (3/8) | 0.000 |
| >3 cm | 71.05% (27/38) | 28.95% (11/38) | |
| LN metastasis | |||
| Negative | 60.0% (9/15) | 40.0% (6/15) | 0.018 |
| Positive | 74.19% (23/31) | 25.81% (8/31) | |
| N1 | 33.33% (1/3) | 66.67% (2/3) | 0.000 |
| N2 | 78.57% (22/28) | 21.43% (6/28) | |
The correlation between TSLP expression and the number of tregs in the tumor microenvironment
Foxp3+ regulatory T cells in tumor microenvironment were detected by immunohistochemistry. The number of Foxp3+ Tregs in lung cancer tissue was significantly increased compared with that in non-cancer lung tissue (P < 0.05). The number of Foxp3+ Tregs was increased in the group that expressed TSLP in tumor tissue, compared with that in the TSLP negative group (P < 0.05), indicating that the expression of TSLP was correlated with the number of regulatory T cells (Fig. 2).
Fig. 2.
Foxp3+ Tregs in tumor tissue and non-cancer lung tissue of lung cancer patients. a Foxp3+ Tregs in tumor tissue and non-cancer lung tissue of lung cancer patients were detected by immunohistochemistry. (200×, →Foxp3+ Tregs). b The number of Foxp3+ regulatory T cells in lung cancer tissue was significantly increased compared with that in non-cancer lung tissue (P < 0.05). The number of Foxp3+ Tregs in cancer tissue that expressed TSLP protein was increased compared with that in the TSLP negative group (P < 0.05)
Effects of TSLP on the phenotype and cytokines secreting level of DCs
The monocytes-derived DCs were stimulated with TSLP or LPS. The phenotype of TSLP-DCs, LPS-DCs, or unstimulated imDCs was examined by flow cytometry. TSLP-DCs expressed high levels of CD86, CD11C, HLA-DR, and low levels of CD14, which were similar to those of the LPS-DCs. TSLP-DCs expressed moderate level of CD83(58.42% ± 19.17%), which was significantly different from imDCs (19.71% ± 7.91%) and LPS-DCs (80.58% ± 11.11%) (Fig. 3a). These results indicated that TSLP-DCs showed the characteristics of intermediate mature DCs.
Fig. 3.
Effects of TSLP on the phenotype and cytokine secretion of dendritic cells. a DCs were induced from PBMCs and left unstimulated or exposed to TSLP or LPS. The phenotype of TSLP-DCs, LPS-DCs, and imDCs was examined by flow cytometry assay. TSLP-DCs expressed high levels of CD86, CD11C, HLA-DR and low levels of CD14, which was similar to LPS-DCs. TSLP-DCs expressed moderate levels of CD83(58.42% ± 19.17%), which showed significant difference compared with imDCs (19.71% ± 7.91%) and LPS-DCs (80.58% ± 11.11%). b The secreted levels of IL-6, IL-10, IL-12p70, IFN-γ, TNF-α, and TGF-β of imDCs, TSLP-DCs and LPS-DCs were analyzed by ELISA method. TSLP-DCs secreted low level of IL-6, IL-10, IL-12p70, IFN-γ, TNF-α, and high level of TGF-β compared with that of LPS-DCs. TSLP-DCs, LPS-DCs, and imDCs secreted high levels of chemokines MDC/CCL22 and TARC/CCL17. (*P imDC versus TSLP-DC, **P TSLP-DC versus LPS-DC)
The secreted levels of IL-6, IL-10, IL-12p70, IFN-γ, TNF-α, and TGF-β of imDCs, TSLP-DCs and LPS-DCs were analyzed by ELISA. LPS-DCs produced high levels of IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α. TSLP-DCs secreted low levels of IL-6, IL-10, IL-12p70, IFN-γ, TNF-α, and high level of TGF-β. Furthermore, because the effects of TSLP-DCs on inducing the migration of Tregs were analyzed in this study, the secreting level of chemokines MDC/CCL22 and TARC/CCL17 was examined. TSLP-DCs, LPS-DCs and imDCs all secreted high levels of chemokines MDC/CCL22 and TARC/CCL17. The secreted level of MDC/CCL22 of TSLP-DCs and LPS-DCs was much higher than that of imDCs (Fig. 3b).
The JAK/STAT signaling pathway in TSLP-DCs
The activation status of the JAK/STAT pathway in dendritic cells stimulated with TSLP or LPS-DC for 24 h and imDC was analyzed using phospho-specific protein microarray. The results indicated that MEK1-Ser217, STAT1-Tyr701, STAT3-Ser727, and STAT5A-Tyr694 were phosphorylated after stimulation with TSLP, which was similar to that observed in imDCs. However, JAK phosphorylation was not observed (Table 2).
Table 2.
JAK/STAT pathway in unstimulated and TSLP- or LPS-stimulated DCs
| Protein list of phospho–non-phospho proteins | TSLP-DCs | imDCs | LPS-DCs |
|---|---|---|---|
| p-JAK1 (Tyr1022)/JAK1 | 0.94 | 0.77 | 1.13 |
| p-JAK2 (Tyr1007)/JAK2 | 0.93 | 0.92 | 0.98 |
| p-JAK2 (Tyr221)/JAK2 | 0.79 | 0.63 | 0.98 |
| p-MEK1 (Ser217)/MEK1 | 1.81 | 1.7 | 1.28 |
| p-MEK1 (Ser221)/MEK1 | 1.17 | 0.85 | 1.15 |
| p-MEK1 (Thr291)/MEK1 | 1.29 | 1.4 | 1.2 |
| p-MEK2 (Thr394)/MEK2 | 1.18 | 1.31 | 1.01 |
| p-ERK (Thr202)/ERK | 1.29 | 1.47 | 1.1 |
| p-ERK (Tyr204)/ERK | 1.51 | 1.93 | 1.32 |
| p-Raf1 (Ser259)/Raf1 | 1.57 | 1.78 | 1.16 |
| p-Raf1 (Ser338)/Raf1 | 1.48 | 1.53 | 1.17 |
| p-STAT1 (Tyr701)/STAT1 | 2.69 | 2.69 | 1.62 |
| p-STAT1 (Ser727)/STAT1 | 1.09 | 1.03 | 1.22 |
| P-STAT3 (Ser727)/STAT3 | 1.82 | 2.13 | 1.55 |
| p-STAT3 (Tyr705)/STAT3 | 1.34 | 1.64 | 1.09 |
| p-STAT4 (Tyr693)/STAT4 | 1.19 | 1.28 | 1.06 |
| p-STAT5A (Tyr694)/STAT5A | 2.35 | 2.12 | 1.61 |
| p-STAT5A (Ser780)/STAT5A | 1.12 | 1.34 | 0.95 |
| p-STAT6(Thr645)/STAT6 | 0.6 | 0.53 | 0.55 |
| p-STAT6(Tyr641)/STAT6 | 1.03 | 1.08 | 0.97 |
| p-TYK2(Tyr1054)/TYK | 1.21 | 1.1 | 1.04 |
To determine whether TSLP directly activated multiple STATs, dendritic cells were stimulated for 15 min with TSLP or LPS. The results of Western blotting indicated that TSLP induced the phosphorylation of STAT1, -3, -5. Contrast to LPS-DCs, the phosphorylation of STAT3 in TSLP-DC was increased (Fig. 4).
Fig. 4.
Activation of STAT proteins in TSLP-DCs, LPS-DCs, or imDCs Western blotting analysis was performed to compare the early time course of activation of STAT proteins in dendritic cells stimulated with TSLP. TSLP induced the phosphorylation of STAT1, STAT3, STAT5 within 15 min. Contrast to LPS-DCs, the activation of STAT3 in TSLP-DC was increased
Effects of TSLP-DCs on the differentiation of CD4+CD25− T cells into CD4+CD25+CD127− T cells
CD4+CD25− T cells were isolated from PBMCs of lung cancer patients and cultured with medium only (non-DCs) or in the presence of TSLP-DCs, LPS-DCs, or imDCs for 5 days at the ratio of 1:10. The percentage of CD4+CD25+CD127− T cells was detected by FACS method. In the presence of TSLP-DCs, the percentage of CD4+CD25+CD127− T cells was (9.70% ± 3.686%), which was statistically higher than those cocultured with imDCs (4.144% ± 0.954%), LPS-DCs (4.563% ± 2.308%), or medium only (2.560% ± 1.138%) (Fig. 5). These data indicated that TSLP-DCs could induce CD4+CD25− T cells differentiate into CD4+CD25+ Tregs.
Fig. 5.
Effects of TSLP on the differentiation of CD4+CD25− T cells into CD4+CD25+CD127− T cells CD4+CD25− T cells were cultured with medium or in the presence of TSLP-DCs, LPS-DCs, or imDCs for 5 days. The percentage of CD4+CD25+CD127− T cells was detected by FACS assay. In the presence of TSLP-DCs, the percentage of CD4+CD25+CD127− T cells was (9.70% ± 3.686%), which was statistically higher than those cocultured with unstimulated DCs (4.144% ± 0.954%), LPS-DCs (4.563% ± 2.308%), or medium only (2.560% ± 1.138%). (*P < 0.05, **P > 0.05)
Effects of TSLP-DCs on the migration of CD4+CD25+ T cells
The effects of TSLP-DCs on the migration of CD4+CD25+ T cells were analyzed by transwell assay. The numbers of CD4+CD25+ T cells migrated into the bottom layer when cocultured with TSLP-DCs and LPS-DCs were significantly higher compared with those cocultured with imDCs (Fig. 6, P < 0.05). This may be the result of the high secretion levels of MDC/CCL22 in the supernatant of both TSLP-DCs and LPS-DCs.
Fig. 6.
Effects of TSLP-DCs on the migration of CD4+CD25+ T cells. a The effects of TSLP-DCs o the migration of CD4+CD25+ T cells were analyzed by transwell assay. The Tregs migrated into the bottom layer of transwell were detected by fluorescence staining of CD25. b The number of CD4+CD25+ T cells migrated into the bottom layer in the coculture with TSLP-DCs was slightly more than that in the coculture with LPS-DCs. (P im-DC versus TSLP-DC < 0.05, P TSLP-DC versus non-DC < 0.05, P TSLP-DC versus LPS-DC > 0.05)
Discussion
Recent reports indicate that CD4+CD25+ Treg cells not only play a central role in controlling autoimmunity but also partake in the inhibition of antitumor immune responses. It has been reported that the CD4+CD25+ Treg cells are increased in the peripheral blood and tumor microenvironment [1–4], suggesting that tumor cells may stimulate the proliferation or differentiation of Tregs. On the other hand, increased Tregs may suppress tumor-specific immune response and play an important role in tumor progression and metastasis. However, the mechanisms underlying the development of Tregs in cancer patients remain unknown.
It has been reported that thymic stromal lymphopoietin is involved in the positive selection of regulatory T cells in thymus and induction of CD4+ T-cell-mediated inflammation [8–10]. TSLP was originally cloned in the conditioned supernatants from a mouse thymic stromal cell line and was known to be expressed by the epithelial cells of Hassall’s corpuscles in humans. TSLP directly activates dendritic cells, inducing the secretion of cytokines and chemokines. Recent studies showed that a subset of DCs activated by TSLP positively select Tregs in the medulla of human thymus [8]. Whether cancer cells secret TSLP and therefore induces Tregs has not been reported previously. In this study, we analyzed whether TSLP participated in the induction of Tregs in cancer patients.
We analyzed the expression of TSLP in tumor tissue and the correlation of TSLP expression with the number of Tregs in the tumor microenvironment. The expression of TSLP protein was significantly increased in tumor tissue compared with that in benign lesion and non-cancer lung tissue. Furthermore, the number of Foxp3+ Tregs in the tumor microenvironment was correlated with the expression of TSLP protein in tumor tissue. However, whether TSLP can induce Tregs remains unclear. To address this, the effects of TSLP on the function of DCs and differentiation/migration of Tregs were further analyzed in vitro.
In contrast to mouse TSLP that stimulates B cells [11], CD4+ T [12] cells and myeloid DCs [13], human TSLP acts mainly on myeloid DCs [14]. The effects of TSLP on the phenotype and cytokine secretion of DCs were detected. It has been reported that TSLP strongly upregulates the expression of MHC class II, CD54, CD80, CD83, and CD86 and DC-LAMP on myeloid DCs allergic inflammation [14]. Here, we found that the TSLP-DCs from lung cancer patients expressed high level of CD11c, CD86, HLA-DR, similar to the DCs stimulated by TSLP during inflammation. However, the expression of CD83 on TSLP-DCs from cancer patients was significantly decreased compared with that on LPS-DCs, but still higher than imDCs, which indicated the characteristics of intermediate stage maturate DCs. Furthermore, TSLP-DCs from lung cancer patients secreted low levels of IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α. It has been reported that TSLP does not stimulate myeloid DCs to produce the TH1-polarizing cytokine IL-12 or the pro-inflammatory cytokines TNF, IL-1β, and IL-6 inflammation [15], which was similar to the TSLP-DCs from lung cancer patients. Moreover, TSLP stimulated DCs from lung cancer patients secreted high level of TGF-β, which could induce the CD4+CD25+ Tregs and participate in antigen-specific immune tolerance [16]. The inability of TSLP to induce the production of Th1-polarizing cytokines and high secreting level of TGF-β in DCs may be related to the function of TSLP-stimulated DCs in the tumor microenvironment.
In order to analyze the mechanisms by which TSLP induces dendritic cells, the activation of JAK/STAT pathway was examined by protein microarray and Western blotting. The results indicated that STAT1, STAT3 and STAT5 were phosphorylated after the stimulation of TSLP for either 15 min or 24 h. However, we did not find the phosphorylation of JAK1 or JAK2. Although it was well established that STAT3/5 were activated by tyrosine phosphorylation through JAK or Src family kinases in response to various cytokines [17, 18], the key events following binding of hTSLP to hTSLPR/IL-7Rα were the activation of STAT3/STAT5 without the phosphorylation of JAK1/JAK3, which indicated that TSLP activated the STAT3/5 through distinct pathways [15].
The influence of TSLP-DCs on the differentiation/migration of Tregs was then examined in vitro. Three possible mechanisms may be involved in the function of TSLP-DCs to induce Tregs: (1) through the expansion of Tregs; (2) through inducing the differentiation of CD4+CD25− T cells into CD4+CD25+ Tregs; and (3) through inducing the migration of Tregs from circulation to the tumor microenvironment. CD4+CD25+ regulatory T cells have been previously identified as anergic cells after T-cell receptor (TCR) stimulation [19]. In this study, the differentiation and migration of regulatory T cells induced by TSLP-DCs were further examined. CD4+CD25− T cells were isolated from lung cancer patients and cocultured with TSLP-DCs, LPS-DCs, or imDCs. The percentage of CD4+CD25+CD127− cells significantly increased in the TSLP-DCs group compared with the other two groups, indicating that TSLP-DCs could induce CD4+CD25− T-cell differentiation into Tregs. It has been reported that TSLP-DCs can promote the differentiation of regulatory T cells from CD4+CD25− naive T cells in thymus [8, 20]. This study for the first time reported the effects of TSLP on the differentiation of Tregs in cancer patients. In addition to this function, the intriguing effect of TSLP-DCs on the migration of Tregs was also evaluated by transwell assay. The results indicated that the number of CD4+CD25+ cells migrated toward TSLP-DCs or LPS-DCs was significantly increased compared to that migrated to imDCs. We also found the secreted levels of chemokine MDC/CCL22 of TSLP-DCs and LPS-DCs were much higher than those of imDCs. CCL22, as one of the chemokines that enhances the migration of FoxP3+ cells, which expresses the CCL22 receptor CCR4 [21, 22], may be involved in the effects on Treg migration.
In conclusion, the expression of TSLP protein was correlated with the number of Tregs in the lung cancer microenvironment. TSLP was involved in the increasing prevalence of regulatory T cells in the cancer microenvironment by inducing the differentiation of CD4+CD25− T cells into Tregs and the migration of Tregs in a DC-dependent manner.
Acknowledgments
This study was supported by National Natural Science Foundation of China (No. 30872986).
Footnotes
Hu Li and Hua Zhao contributed equally to this manuscript.
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