Suppressive effects of gemcitabine plus cisplatin chemotherapy on regulatory T cells in nonsmall-cell lung cancer

Abstract

Objective

To investigate the effect of gemcitabine plus cisplatin chemotherapy on the percentage of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– regulatory T cells (Tregs) in the peripheral blood of patients with nonsmall-cell lung cancer (NSCLC).

Methods

Peripheral blood was taken from patients with NCSLC (before and after chemotherapy) and control subjects with nonmalignant disease. The percentages of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Tregs were analysed using flow cytometry.

Results

Patients (n = 40) had significantly higher CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– percentages than control subjects (n = 24). CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– percentages increased with tumour progression, fell significantly after chemotherapy, but remained significantly higher than control values.

Conclusions

CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Treg percentages were higher in patients with NSCLC than control subjects, and increased in line with tumour progression. Percentages of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Tregs were significantly reduced following gemcitabine plus cisplatin chemotherapy.

Keywords

chemotherapy NSCLC

Introduction

Immunosuppressive regulatory T cells (Tregs) play critical roles in controlling cancer immune evasion.^1,2 Tregs belong to several lineages including CD4⁺CD25⁺FOXP3⁺ T cells, which inhibit antitumour immunity by suppressing tumour-specific effector T cells. These Tregs function through cell-to-cell interactions³ and/or secretion of cytokines such as interleukin (IL)-10 or transforming growth factor (TGF)-β.^4,5 The number and activity of CD4⁺CD25⁺FOXP3⁺ Tregs increase in patients with cancer,^1,6–10 and their presence correlates with unfavourable prognosis. CD8⁺CD28^– Tregs also have a regulatory function,^10,11 suppressing the cytotoxic function and proliferation of T lymphocytes. It is possible that the depletion or suppression of Tregs could prevent tumour immune escape.^12,13

Chemotherapy cannot discriminate between neoplastic and non-neoplastic cells, and may therefore be detrimental to host-antitumour immunity. Conventional chemotherapeutic agents (including cyclophosphamide and fludarabine) downregulate the number and function of CD4⁺CD25⁺ Tregs in animal models¹⁴ and human cancer.¹⁵ First-line treatment for nonsmall-cell lung cancer (NSCLC) is gemcitabine plus cisplatin chemotherapy, but little is known regarding the immunomodulatory effects of this treatment. Gemcitabine enhances antitumor immune activity by selectively eliminating splenic Gr-1⁺/CD11b⁺ myeloid suppressor cells in tumour-bearing animal models.¹⁶ In addition, cisplatin plus vinorelbine augment antitumor immunity by modulating the function and reducing the proportion of CD4⁺CD25⁺FOXP3⁺ Tregs in animal models.¹⁷ It is not known whether the therapeutic doses of gemcitabine plus cisplatin would produce similar results in patients with NSCLC. In addition, there are no data available regarding the sensitivity of CD8⁺CD28^– Tregs to cisplatin or gemcitabine.

The aim of this study was to investigate the effect of gemcitabine plus cisplatin chemotherapy on the percentage of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– regulatory T cells (Tregs) and natural killer (NK) cells in the peripheral blood of patients with NSCLC.

Patients and methods

Study population

The study included patients aged >20 and <75 years with histologically confirmed NSCLC who were treated at Zhoushan Hospital, Zhoushan, China between October 2012 and July 2013. Tumours were staged according to NCCN clinical practice guidelines (2012).¹⁸ The control group comprised patients with nonmalignant disease. Inclusion criteria were: Karnofsky performance status (PS) >80%; satisfactory haematological and biochemical parameters (neutrophil count ≥2.0 × 10⁹/l, haemoglobin ≥100.0 g/l, platelet count ≥100 × 10⁹/l, aspartate aminotransferase [AST] and alanine aminotransferase [ALT] ≤80U/l); life expectancy >3 months. Exclusion criteria were: previous malignancies; secondary lung cancer; previous anticancer treatment; concomitant immune enhancing or immune suppressing diseases including bacterial, fungal or viral infections.

The study was approved by the ethics committee of Zhoushan Hospital, Zhoushan, China, and written informed consent was obtained from all participants.

Patient assessment and treatment

Before treatment, patients underwent physical examination, chest computed tomography (CT), head CT or magnetic resonance imaging (MRI), electrocardiogram, abdominal B-ultrasonography, pulmonary function tests, emission computed tomography, full blood count and serum biochemical analyses.

Chemotherapy was a 21 = day treatment cycle comprising gemcitabine 1250 mg/m² on days 1 and 8, and cisplatin 75 mg/m² on day 1.

Flow cytometry

Peripheral blood (2 ml) was taken before surgery, on day 0 (before initiation of first round of chemotherapy) and day 20 (before initiation of second round of chemotherapy). Blood samples were collected in sterile heparinized vials and analysed immediately. Aliquots of heparinized whole blood (100 µl) were incubated for 15 min at room temperature with antibodies (all BD Biosciences, San Jose, CA, USA): (i) FITC-conjugated mouse antihuman CD4, APC-conjugated mouse antihuman CD25 and PE-conjugated mouse antihuman FOXP3; (ii) FITC-conjugated mouse antihuman CD8 and PE-conjugated mouse antihuman CD28; or (iii) FITC-conjugated mouse antihuman CD3 and PE-conjugated mouse antihuman CD16/CD56. Erythrocytes were lysed using lysing solution and washed twice with phosphate-buffered saline (pH 7.4), containing 2% fetal calf serum and 2% sodium azide. Intranuclear staining of FOXP3 was performed according to the manufacturer’s instructions (BD Biosciences).

Cells were analysed by FACSCalibur™ (BD Biosciences), and the resulting data were analysed using CellQuest™ software. A minimum of 5000 gated events/condition were analysed. CD4⁺CD25⁺FOXP3⁺ cells were quantified as a percentage of CD4⁺ cells, CD8⁺CD28^– cells were quantified as a percentage of CD8⁺ cells, and NK (CD3^–CD16⁺CD56⁺) cells were quantified as a percentage of lymphocytes.

Statistical analyses

Date were expressed as mean ± SD. Due to nonhomogeneity of variance and small sample size, data comparing differences between patients and controls in Treg percentages were assessed using Mann–Whitney nonparametric U-test. The differences between tumour subgroups and controls in the Treg percentages were analysed using one-way analysis of variance (ANOVA) followed by LSD for multiple mean comparisons. When variables were not normally distributed, Kruskal–Wallis test followed by Dunnett T3 was used. Comparisons of Treg percentages before and after chemotherapy were made using paired-sample t-test. Statistical analyses were performed using SPSS® version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values <0.05 were considered statistically significant.

Results

The study included 40 patients with NSCLC (30 male/10 female; mean age 62.1 ± 9.2 years; age range 38–74 years) and 24 control subjects (17 male/7 female; mean age 57.8 ± 14.3 years; age range 35–74 years). There were no statistically significant between-group differences in age or sex. In the patient group, four individuals with stage Ia tumours underwent surgery only, and seven patients with stage IV tumours received chemotherapy only. The remaining 29 patients (stage Ib–IIIa) underwent surgery followed by chemotherapy 30 days later. Tumours included squamous carcinoma (n = 17) and adenocarcinoma (n = 23).

In samples taken before surgery, patients with NSCLC had significantly higher CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Treg percentages than control subjects (P < 0.001 for each comparison; Table 1). There was no between-group difference in percentages of NK cells.

Table 1.

Percentages of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– regulatory T cells and natural killer (NK; CD3^–CD16⁺CD56⁺) cells in peripheral blood of patients with nonsmall-cell lung cancer, stratified according to tumour stage, and control subjects with nonmalignant disease.

Cell type	Control group n=24	Patient group
Cell type	Control group n=24	Total n = 40	Stage I/II n = 21	Stage III/IV n = 19
CD4⁺CD25⁺FOXP3⁺/CD4⁺, %	1.44 ± 0.66	3.26 ± 1.43 ^a	2.4 ± 8.84 ^b,c	4.21 ± 1.36 ^b,c
CD8⁺CD28^–/CD8⁺, %	36.70 ± 8.31	56.40 ± 14.39 ^a	48.61 ± 11.37 ^d,e	65.02 ± 12.50 ^d,e
NK cells/lymphocytes, %	19.38 ± 6.40	23.06 ± 10.94	NA	NA

Data presented as mean ± SD.

P < 0.001 versus control group; Mann–Whitney U-test.

P < 0.001 within subgroups; Dunnett T3 test.

P < 0.001 versus control group; Kruskal–Wallis test.

P < 0.001 within subgroups; one-way analysis of variance.

P < 0.001 within subgroups; LSD test.

NA, data not available.

NK, natural killer cells (CD3^–CD16⁺CD56⁺).

Patients with NSCLC were stratified according to tumour stage (stage I/II and stage III/IV). Percentages of both CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Tregs increased significantly with tumour progression (P < 0.001 for each comparison; Table 1).

Populations of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Tregs were significantly lower after chemotherapy than at baseline in the total patient group (P < 0.001 for each comparison; Table 2). There were no significant changes in percentages of NK cells. Postchemotherapy percentages of CD4⁺CD25⁺FOXP3⁺ Tregs were significantly higher in patients with stage III/IV disease than in controls (P < 0.001; Table 2). Postchemotherapy percentages of CD8⁺CD28^– Tregs were significantly higher in both patient subgroups than in controls (P < 0.001 for each comparison, Table 2).

Table 2.

Cell type	Control group n = 24	Patient group
Cell type	Control group n = 24	Total n = 36	Stage I/II n = 17	Stage III/IV n = 19
CD4⁺CD25⁺FOXP3⁺/CD4⁺, %
Baseline	1.44 ± 0.66	3.85 ± 1.98	2.57 ± 1.41	4.99 ± 1.72
After chemotherapy	–	2.27 ± 1.11^a	1.80 ± 0.92^b	2.68 ± 1.12^{b c}
CD8⁺CD28^–/CD8⁺, %
Baseline	36.70 ± 8.31	63.09 ± 10.89	59.61 ± 11.25	66.19 ± 9.82
After chemotherapy	–	53.24 ± 9.74^a	51.06 ± 8.75^d	55.20 ± 10.39^d
NK cells/lymphocytes, %
Baseline	19.38 ± 6.40	26.53 ± 10.97	NA	NA
After chemotherapy	–	27.93 ± 12.36	NA	NA

Data presented as mean ± SD.

P < 0.001 vs baseline; paired-sample t-test.

P < 0.001 vs baseline; Kruskal–Wallis test.

P < 0.001 vs control group baseline; Dunnett T3 test.

P < 0.001 vs control group baseline; one-way analysis of variance.

NA, data not available.

NK, natural killer cells (CD3^–CD16⁺CD56⁺).

Discussion

Percentages of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Tregs were higher in patients with NSCLC than in control subjects with nonmalignant lung disease in the present study. In addition, CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Treg populations increased with tumour progression, which is consistent with findings from other cancer types.^{10,11,19–21} In accordance with findings in early stage prostate cancer,⁸ the present study indicated a higher percentage of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Tregs in early stage NSCLC patients compared with control subjects.

It is thought that Tregs may play a crucial role in inhibiting anticancer defence, and their depletion or suppression may therefore represent an important strategy to prevent tumour immune escape.^12,22 Chemotherapy was traditionally thought to be deleterious to antitumour immunity, but some chemotherapy agents may have the potential to augment anticancer immunity by inhibiting immune tolerance and suppression. Paclitaxel has been found to inhibit the number and function of CD4⁺CD25⁺FOXP3⁺ Tregs directly.²³ In addition, cyclophosphamide was reported to inhibit the activity of CD4⁺CD25⁺ Tregs,¹⁴ and the generation and function of CD8⁺CD28^– Tregs.²⁴ In contrast, arsenic trioxide, dacarbazine, 5-fluorouracil and methotrexate increase the frequency of Tregs in peripheral blood mononuclear cells activated with T-cell mitogen.²⁵ The above data suggest that chemotherapeutic agents may have varying effects on Treg populations and activity.

Percentages of CD4⁺CD25⁺FOXP3⁺ and CD8+CD28^– Tregs were significantly reduced following chemotherapy in the present study. To our knowledge, this is the first study to focus on changes in Treg subpopulations in the peripheral blood of patients with NSCLC undergoing gemcitabine plus cisplatin chemotherapy. Our findings suggest that, in addition to inducing tumour cell death via cytotoxic effects, gemcitabine plus cisplatin exert anticancer activity by decreasing the numbers of circulating Tregs in these patients. The fact that CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Treg postchemotherapy percentages remained higher than those of control subjects suggests that the remaining cancer may continue to feed the mechanism underlying the raised Treg levels.

The present study has several limitations. The study population was small, necessitating the merging of subgroups for statistical analyses. As a result our findings should be interpreted cautiously, since a larger sample size is needed. In addition, it was outside the scope of our study to determine whether the CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– cells we identified displayed suppressive activity. Consequently we cannot categorically state that these cells are regulatory/suppressive T cells. Chemotherapy may not only decrease Treg number but also inhibit functionality,^23,26 and further investigations into functional changes will allow insight into the immunoregulatory effect of gemcitabine plus cisplatin chemotherapy. Our study investigated combination gemcitabine and cisplatin chemotherapy, and it is not possible to extrapolate our findings to determine the impact of each agent individually. This could be overcome via dose–response analysis of single agents in animal models. Finally, NK cells provide a first line of defence against infections and malignant cells, but our study found no significant differences in NK-cell percentages between patients and controls, as well as before and after chemotherapy. NK cells have been reported to be dysfunctional in a number of cancers,^27–29 and to be affected in various ways by exposure to chemotherapeutic agents.^30–34 Further studies are required to investigate the function of NK cells during chemotherapy, and to explain more fully the relationship between chemotherapy and antitumour immunity.

In conclusion, CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Treg percentages were higher in patients with NSCLC than control subjects with nonmalignant lung disease, and increased in line with tumour progression. Percentages of CD4⁺CD25⁺FOXP3⁺ and CD8⁺CD28^– Tregs were significantly reduced following gemcitabine plus cisplatin chemotherapy.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research was supported by the Science and Technology Program of Zhoushan (No.2012B17).

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