Abstract Objective To investigate the Gemzar plus cisplatin for non-small cell lung cancer patients with immune tolerance role. Methods 38 cases of pathologically or cytologically confirmed NSCLC patients with gemcitabine and cisplatin combination chemotherapy regimens, and analyzed by flow cytometry before and after chemotherapy, peripheral blood CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in CD4 + T-cell percentage . Results of chemotherapy before NSCLC patients with peripheral blood CD4 + CD25 + Foxp3 + regulating T cells (Treg) accounted for CD4 + T cells in the ratio was significantly higher than the healthy control group (P <0.05); chemotherapy after NSCLC patients peripheral blood CD4 + CD25 + Foxp3 + regulating T cells (Treg ) in CD4 + T cell percentage is higher than before chemotherapy was significantly lower (P <0.05); squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma among the three groups before chemotherapy, after chemotherapy, the differences between the various indicators not statistically significant (P> 0.05). Conclusion Gemzar plus cisplatin chemotherapy may be the body’s regulation of advanced non-small cell lung cancer tumor immune tolerance, improve the patient’s immune function.

Key words CD4 + CD25 + Foxp3 + regulatory T cells (Treg) flow cytometry chemotherapy

   Lung cancer is a common malignant tumors of the serious threat to human life and health, it’s happening, development and immune function status. Cellular immunity is an important mechanism for the anti-tumor immune-specific cellular immune T cell-mediated cellular immune response. T cells are important immunoregulatory cells, may play a positive and negative regulatory role [1]. In the mid-nineties of the last century, was first confirmed by Sakaguchi et CD4 CD25 regulatory T cells (regulatory T cell, Treg), a class of T cells has immunosuppressive effects, accounting for the outer periphery of CD4 T cells from 5% to 10 %. Not only can inhibit the occurrence of autoimmune diseases, may also be involved in the regulation of tumor immunity, tumor environment CD4 + CD25 + Treg increase in the proportion of to cause tumor immune tolerance [2]. Most of the non-small cell lung cancer (non  small cell lung cancer, NSCLC) diagnosed are late, chemotherapy has become an important treatment. Gemcitabine (Gemzar Gemcitabine chemical name gemcitabine hydrochloride) is a new type of synthetic pyrimidine nucleotide analogues. The information shows that Gemzar is a good effect, a new anticancer drug with fewer side effects. Cisplatin (Gemcitabine + Cisplatin, GP) is the first-line treatment of lung cancer have significant clinical efficacy. “GP" regimen of CD4 + CD25 + regulatory T cells, foreign rare reports; domestic been reported.

    In this paper, flow cytometry analysis of 38 patients with NSCLC peripheral blood CD4 + CD25 + Foxp3 + Treg cell percentage in the gemcitabine plus cisplatin chemotherapy before and after the change, so as to explore the impact of chemotherapy on the immune function of patients with NSCLC.

1 Materials and Methods

    1.1 The object of study

    The group of 38 patients with primary NSCLC patients are inpatients admitted I oncology centers from January 2006 to July 2007, aged 37 to 72 years, mean age 55.1 ± 7.2 years, including 25 males and 13 females. The naive 28 cases, retreatment were 10 cases. According to the the TNM method proposed UICC1992 installments divided into seven cases: Phase IIIa, Ⅲ b of 10 cases, Ⅳ of 21 cases. Pathological type: 14 cases of squamous cell carcinoma, 21 adenocarcinoma, adenosquamous carcinoma in three cases. 38 cases were confirmed by pathology or cytology; measurable lesions; Karnofsky score> 60; expected survival ≥ 3 months, normal liver and kidney function; within one month before treatment is not done other anti- tumor treatment, nearly three months unused immunostimulants. The control group of 15 cases of hospital healthy subjects, 8 males, 7 females, age 26 to 68 years, with an average of 48.2 ± 12 years.

    1.2 Research Design and chemotherapy

    Before chemotherapy, three days after the end of a week of chemotherapy collecting peripheral blood of patients with NSCLC, detection of CD4 + CD25 + Foxp3 + regulatory T cell levels. GP regimen of chemotherapy regimens are used: Gemzar 1250mg/m2 intravenous infusion 30min drops finished, d1, 8; cisplatin 30mg/m2 intravenous infusion, d1 ~~ d3; 21d for 1 cycle.

    1.3 CD4 + CD25 + Foxp3 + regulatory T cells detected

    1.3.1 reagents and equipment FITC-labeled mouse anti-human CD3 antibody, PE-labeled mouse anti-human CD4 antibody, PE-labeled mouse anti-human CD8, FITC-labeled mouse anti-human CD25 antibody, Cychrome-labeled CD69 and CD45RA antibody, PE -labeled mouse anti-human Foxp3 antibody. CD4 isotype controls were PE-labeled mouse anti-human IgG1 immunoglobulin, FITC-labeled mouse anti-human IgG1 immunoglobulin (all purchased from BD Biosciences, USA).

    1.3.2 Flow cytometry analysis of all observed objects are in the early morning fasting blood samples were 2 ml of EDTA anticoagulant. Fluorescent direct labeling method and flow cytometry in peripheral blood mononuclear cells (PBMC) membrane surface CD3, CD4, CD8, CD25. Different fluorescently labeled monoclonal antibodies each 20μl 100μl heparinized peripheral blood were added and incubated at room temperature for 15min, then added erythrocyte lysis buffer PBS was washed 2 times with a flow cytometer (BD Biosciences, FACS Calibur) detection Cellquest software analysis data, record the percentage of positive cells subtracting nonspecific control values.

    Foxp3 detection: peripheral blood heparin, 1500r/min centrifugal 5min, the supernatant was removed, the cells were collected, the collected cells were resuspended in PBS. Then take two tubes are labeled 1 and 2, a plus CD4  percp 3.5μl, 2 plus CD4  percp CD25  FITC 3.5 μL, and then incubated for 20min, 150μl of PBS was added, further added 1 ml of red blood cell lysis buffer to avoid light to 10min, 1500r/min centrifugal 5min, the supernatant was removed and the precipitate resuspended in 1ml PBS 1500r/min centrifugal 5min, the supernatant was removed by adding Foxp3 breaking film former 500 μl of the dark at room temperature 30min. 1500r/min centrifugal 5min, the supernatant was precipitated 1ml PBS the resuspended (repeat). 2 plus 7μl Foxp3  PE, then 1 and 2 at 4 ℃ for 30 min. Add 1ml PBS, resuspended again 1500r/min centrifugal 5min, the supernatant was removed. Add 1ml PBS, resuspended again 1000r/min centrifugal 5min, the supernatant was removed. Add 300μl PBS resuspended detected on the machine.

    1.4 Statistical Methods SPSS13.0 statistical software t test and analysis of variance. P <0.05 was considered statistically significant.

2 Results

    2.1 lung peripheral blood CD4 + CD25 + Foxp3 + Treg cell ratio increased

    Healthy controls peripheral CD4 + CD25 + Foxp3 + Treg in CD4 + T cell ratio was 0.51% ± 0.54%; patients with lung cancer in peripheral blood CD4 + CD25 + Foxp3 + Treg in CD4 + T cell ratio was 1.96% ± 0.73%, two group, the difference was statistically significant (P <0.05), as shown in Figure 1 and 2.

    2.2 Gemzar plus cisplatin chemotherapy of NSCLC patients with peripheral blood CD4 + CD25 + Foxp3 + Treg’s impact

    Compared with chemotherapy before and after chemotherapy, the CD4 + CD25 + Foxp3 + / CD4 + ratio was significantly lower than that before chemotherapy, the difference was statistically significant (P <0.05), as shown in Table 1.

    Figure 1 normal control (B  C) and NSCLC (D  E) streaming representatives Figure (A experimental negative control) cellular level of total CD4T cell ratio and the normal control group Table 1 gemcitabine plus cisplatin chemotherapy for NSCLC peripheral blood of patients

2.3 Gemzar plus cisplatin chemotherapy peripheral blood CD4 + CD25 + Foxp3 + Treg the impact of different types of NSCLC patients

    ① before chemotherapy, squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma patients of CD4 + CD25 + Foxp3 + / CD4 + cell ratio was significantly higher than that in the control group (P <0.05); ② three groups after chemotherapy in patients with CD4 + CD25 can be seen from Table 2: + Foxp3 + / CD4 + cell ratio was significantly lower than before chemotherapy (P <0.05); ③ The three groups before chemotherapy and after chemotherapy, the differences between the various indicators was not statistically significant (P> 0.05). Table 2 gemcitabine plus cisplatin chemotherapy # compare with chemotherapy before the peripheral blood of patients with different types of NSCLC (compared to pre  chemotherapy group): P <0.05

3 Discussion

    The occurrence of lung cancer development and prognosis of the immune function of the state, especially the cellular immune function are closely related. Can be expressed CD25 + (ie, the IL-2 receptor a chain), CD4 + T cells are found in recent years, a new type of immune suppressor cells, play an important negative regulatory role in the immune response can be lowered target cells IL  2Ra chain expression, inhibition of proliferation of target cells. In recent years, studies have shown that Treg-mediated immune suppression is one of the important mechanisms of tumor immune escape, is also a major obstacle for tumor immunotherapy [3,4]. A lot of experimental evidence that one of the important mechanisms of tolerance established by the CD4 + CD25 (+) Treg cell-mediated regulation of autoreactive cells autoimmune. Therefore, we can speculate that the CD4 + CD25 + Treg cells while maintaining self-tolerance may also inhibit tumor immunity. Removal of CD4 + CD25 + Treg cells can promote the body of the tumor cells express a variety of antigens [5]. Foxp3 is currently recognized as the CD4 + CD25 + of Treg cells specific markers. Closely related to the expression and function of CD4CD25 regulatory T cells, which play a very important role in the maturation and function of regulatory T cells, only Foxp3 + CD4 + CD25 + of Treg cells before immunosuppressive function [6,7].

    Our previous studies have shown that increase in the proportion of regulatory T cells, and III and IV patients with peripheral blood CD4 + CD25 + of Treg level high of NSCLC patients with peripheral blood CD4 + CD25 + of Treg proportion increase, suggesting that the peripheral blood of malignant tumors in patients with immune dysfunction and cancer patients Ⅰ, Ⅱ patients [8], suggesting that patients with NSCLC immune function is suppressed. Simultaneous detection of peripheral blood CD4 + CD25 + Treg level may progress to prompt the malignancy. The mechanism of CD4 + CD25 + Treg cells how to suppress effector T cells is not entirely clear. Animal experiments confirmed the application of anti-CD25 antibody inhibition of CD4 + CD25 + Treg function can improve anti-tumor immunity [9,10]

    Currently, 70% of NSCLC patients have lost the chance of operation, and thus become one of its main treatment method chemotherapy. Explore the impact of chemotherapy on immune function and immune function and tumor treatment effect relationship has important clinical significance for improving the level of comprehensive cancer treatment to enhance understanding of the prognosis. Gemzar as a first-line treatment for lung cancer chemotherapy is widely used in clinical, mainly through the inhibition of tumor cell DNA synthesis, cells were blocked at the G1 phase, eventually leading to tumor cell apoptosis and exert its cytotoxic effect, is a cell cycle-specific antineoplastic agents. Gemzar plus cisplatin chemotherapy efficiency between 29% to 54% [11].

    In this study show that: with advanced NSCLC patients applications Kin Optional cisplatin regimen after chemotherapy in peripheral blood CD4 + CD25 + of Foxp3 + / CD4 + cell ratio than before chemotherapy significantly with lower (P <0.05), after chemotherapy, and the control group close; Description GP regimen chemotherapy can be cut the Treg level, which prompted the NSCLC patients in the application of Gemzar plus cisplatin regimen not only by the cytotoxicity effective control of tumor, tumor environment can down the Treg levels of confrontation tumor immune escape effectively improve the anti-tumor immune function, improve prognosis. The study provides a new theoretical basis results for the GP program Select Initial treatment of advanced NSCLC. The study also showed before and after chemotherapy with advanced NSCLC, squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma among the three groups of different types of peripheral blood CD4 + CD25 + Foxp3 + / CD4 + ratio difference was not statistically significant (P> 0.05), need to be further expanding the number of cases, stratified analysis. In future studies of the comparative study of GP regimen with other chemotherapy of NSCLC patients peripheral blood CD4 + CD25 + Foxp3 + Treg cells impact, as well as the changes in the characteristics of the different time points after chemotherapy, and discuss its clinical significance.