OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2017.6146 OL-0-0-6146 Articles Functional analysis of CD14+HLA-DR−/low myeloid-derived suppressor cells in patients with lung squamous cell carcinoma ChenYun 1 PanGuichang 2 TianDongbo 2 ZhangYifei 2 LiTaoping 1 Department of Sleep Medicine Center, NanFang Hospital of Southern Medical University, Guangzhou, Guangdong 510515, P.R. China Department of Respiration, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, Guangdong 511500, P.R. China Correspondence to: Dr Taoping Li, Department of Sleep Medicine Center, NanFang Hospital of Southern Medical University, 1838 North Guangzhou Road, Guangzhou, Guangdong 510515, P.R. China, E-mail: toupaper@yeah.net 07 2017 10 05 2017 14 1 349 354 20012016 23022017 Copyright © 2017, Spandidos Publications 2017

Immunomodulatory therapy is a potential effective treatment for advanced cancer that may provide an alternative to chemotherapy, which patients can experience adverse side effects to. Myeloid-derived suppressor cells (MDSCs) have been demonstrated to cause T-cell tolerance in rodents and humans; however, little is known about the role of MDSCs in squamous cell carcinoma. In the present study, the role of MDSCs in lung squamous cell carcinoma was investigated. Peripheral blood from 78 patients with lung squamous cell carcinoma and 30 healthy controls was examined for the presence and function of human MDSCs, denoted as monocyte differentiation antigen CD14-positive HLA class II histocompatibility antigen DR-negative/low (CD14+ HLA-DR−/low) cells by flow cytometry. The sorted T-cell surface glyoprotein CD3 (CD3)+ cells and CD14+HLA-DR−/low cells were subsequently co-cultured with a tumor cell line (NCI-H226). T-cell apoptosis was detected using annexin-V-fluorescein isothicyanate and 7-aminoactinomycin D. Interferon-γ (IFN-γ) levels were detected using an ELISA. The frequency of MDSCs in the peripheral blood mononuclear cells (PBMCs) from patients with lung squamous cell carcinoma was significantly higher compared with that of the healthy controls (P<0.05), whereas the frequency of T-cell surface glyoprotein CD4 (CD4)+ T cells and CD8+ T cells in PBMCs was significantly decreased (P<0.05). In an MDSC/CD8+ co-culture system, the proportion of CD8+ T-cell apoptosis significantly increased with the increase in ratio of MDSCs (P<0.05), while the proportion of tumor cell apoptosis significantly decreased (P<0.05). The concentration of IFN-γ significantly decreased with the increase in MDSCs (P<0.05). Therefore, MDSCs participate in the immune escape of lung squamous cell carcinoma, and may provide a possible therapeutic strategy for the treatment of this disease.

 lung squamous cell carcinoma immunomodulatory therapy myeloid-derived suppressor cells oncology immunology Introduction

Lung cancer is the most common cancer worldwide, with estimates revealing that almost half of all new lung cancer cases occur in Asia, the majority of them in China. Due to the high prevalence of smoking in China, the rate of lung cancer is higher than that of the majority of European and American countries (1). In addition, due to the high prevalence of smoking, ~30% of lung cancer diagnoses are classified as the squamous histopathological subtype (2). In total, ~80% of patients with lung cancer in China exhibit metastases either at the time of presentation or later in the course of the disease, leading to a high mortality rate (3).

Myeloid-derived suppressor cells (MDSCs), a type of immunosuppressive cell, have previously been demonstrated to serve a role in carcinoma (4). Human MDSCs are a heterogeneous population composed of cells at several differentiation stages of the myeloid lineage (5). Different types of tumors harbor distinct subsets of MDSCs, which can be further divided into granulocytic cluster of differentiation antigen 15-positive HLA class II histocompatibility antigen DR-negative/low (CD15+HLA-DR−/low) and monocytic CD14+HLA-DR−/low monocytic MDSC subsets (6). A recent study identified the existence of a monocytic subset of MDSCs with the CD14+HLA-DR−/low phenotype that suppresses the proliferation of T cells (7).

The purpose of the present study was to investigate the proportion of peripheral CD14+HLA-DR−/low MDSCs in patients with different stages of lung squamous cell carcinoma, and to investigate the association between different tumor stages and MDSC function.

 Materials and methods <sec> <title>Patients and healthy donors

A total of 78 patients (67 male and 11 female) diagnosed from January 2014 to October 2015 with lung squamous cell carcinoma at NanFang Hospital of Southern Medical University (Guangzhou, China) were enrolled. The patients were aged between 48 and 72 years old (mean, 58.4 years old). The diagnosis and stage classification of these patients were performed according to the American College of Chest Physicians guidelines released in 2013 (8,9). None of the patients had received chemotherapy or surgery prior to the blood sample being taken. Patients with autoimmune diseases, infectious diseases, multi-primary cancers and other serious diseases were excluded from the current study. All patients were divided into four stages according to the tumor-node-metastasis (TNM) diagnostic criteria (10). Among them, there were 0 patients with stage I, 15 patients with stage II, 37 patients with stage III and 26 patients with stage IV lung squamous cell carcinoma. As the healthy control, 30 healthy volunteers were enrolled in the current study. Blood samples were collected from the aforementioned patients and healthy controls. The current study was approved by the Ethics Committee of NanFang Hospital of Southern Medical University (Guangzhou, China). Written informed consent was obtained from each patient and healthy donor.

 Cell isolation and sorting

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples using Ficoll-Hypaque density gradient centrifugation at 2,500 × g for 20 min at 22°C. MDSCs were isolated from the PBMCs using Miltenyi Macs kit for CD14+ and HLA-DR− (cat. no. 130-091-632; Miltenyi Biotech, Inc., Cambridge, MA, USA), according to the manufacturer's protocol, followed by analysis using a BD FACSAria™ cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). The purity of the MDSCs was >90%, which was derived using flow cytometry software FlowJo 7.6.1 (FlowJo LLC, Ashland, OR, USA). The CD3+ T cells were separated from the PBMCs via CD3+ selection using a MidiMACS™ separator unit (Miltenyi Biotech, Inc.), according to the manufacturer's protocol. The purity of the CD3+ T cells was >95%.

 Flow cytometryto determine the frequency of CD14<sup>+</sup>HLA-DR<sup>−/low</sup> cells in PBMCs from patients

Multicolor fluorescence-activated cell sorting (FACS) analysis was performed using the following antibodies: Anti-CD14 (560634, 20 µg/ml), anti-HLA-DR-PerCp (552764, 10 µg/ml), anti-CD3-APC (565119, 5 µg/ml), anti-CD4-PE (562281, 20 µg/ml) and anti-CD8-FITC (555366, 20 µg/ml), all supplied by BD Pharmingen, San Diego, CA, USA. Flow cytometry was performed using a FACS Calibur™ flow cytometer (BD Biosciences), according to a previously descibed method (11). Analysis of the FACS data was performed using FlowJo software (version X.0.7; TreeStar, Inc., Ashland, OR, USA). Isotype-matched antibodies were used with all the samples as controls.

 Apoptosis assay

The CD4+ and CD8+ T cells were co-cultured with MDSCs in the upper compartment of a Transwell plate (EMD Millipore, Billerica, MA, USA) at different ratios (10,000:0, 10,000:1,000, 10,000:5,000, 10,000:10,000 cells) and treated with monoclonal antibodies anti-CD3 (catalog no. 555337; BD Biosciences; 10 µg/ml) for 48 h at 37.0°C and anti-CD8 (catalog no. 557084; BD Biosciences; 20 µg/ml) for 48 h at 37°C. The same proportions of NCI-H226 tumor cells (10,000 cells/well) were cultured in the lower compartment of the Transwell plate at 37°C (Shanghai Shun Biotechnology, Shanghai, China). Following incubation for 48 h, the cells were collected and stained with annexin-V-fluorescein isothicyanate and 7-amino-actinomycin D (eBioscience, Inc., San Diego, CA, USA), respectively. CD3+ cells were stained with anti-CD8-PerCp (catalog no. 560662; 0.5 mg/ml; BD Biosciences) for 20 min at 20°C to analyze the apoptosis of CD8+ cells. IFN-γ in the supernatant was tested using an ELISA kit (RapidBio Laboratory, Calabasas, CA, USA), according to the manufacturer's protocol.

 Statistical analysis

All statistical analyses were performed using SPSS software (version 6; SPSS, Inc., Chicago, IL, USA). Comparisons between different groups were analyzed using a Mann-Whitney U test. All data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Patient clinicopathological characteristics

The clinicopathological characteristics and clinical stage of the 78 patients and 30 healthy controls are illustrated in Table I. There was no significant difference between the age and gender of the study subjects in the control group and the lung squamous cell carcinoma group. All patients were diagnosed with lung squamous cell carcinoma by clinical methods, imaging, bronchoscopy and pathology. The mean levels of squamous cell carcinoma (SCC) antigen and carcinoembryonic antigen (CEA) in the patients with lung squamous cell carcinoma were 23.8±1.5 µg/l and 135.9±34.1 ng/l, respectively. The normal reference range of SCC is ≤1.5 µg/l and the normal reference range of CEA is ≤5 ng/ml.

 Frequency of MDSCs is significantly increased in patients with lung squamous cell carcinoma compared with that of healthy controls

MDSC frequency in the peripheral blood was analyzed using flow cytometry following density gradient centrifugation. To exclude debris and dead cells, the lymphocytes were selected. Next, CD14+ cells were selected, followed by gating of the HLA-DR−/low population (Fig. 1A). The frequency of MDSCs in the peripheral blood of patients with lung squamous cell carcinoma was significantly higher compared with that of healthy controls (5.38±0.52 vs. 7.664±0.38%; P=0.0014; Fig. 1B).

 Frequency of MDSCs positively correlates with disease stage in patients with lung squamous cell carcinoma

In order to further reveal the role of MDSCs, the TNM staging method, which is based on tumor size, lymph node metastasis, tumor-localized metastasis and tumor-distant metastasis, was used to stage each patient. The frequency of MDSCs was positively correlated with TNM stage (Fig. 2A). The frequencies of MDSCs in patients with stage II, III and IV lung squamous cell carcinoma were 6.51±3.61, 6.51±2.97 and 6.82±3.45%, respectively (P=0.0055; Fig. 2A).

 Frequencies of CD4<sup>+</sup> T cells and CD8<sup>+</sup> T cells in PBMCs from patients with lung squamous cell carcinoma are significantly decreased compared with those of healthy controls

To further investigate the different functions of the MDSCs in patients and healthy controls, the percentages of circulating CD4+ T cells and CD8+ T cells were measured (Fig. 2B and C). The percentages of CD4+ T cells and CD8+ T cells in the PBMCs of patients with lung squamous cell carcinoma were significantly decreased compared with those of healthy controls (28.97±1.51 vs. 25.71±0.83%, P=0.0484; and 15.20±1.31 vs. 11.84±0.85%, P=0.0377, respectively; Fig. 2B and C).

 MDSCs inhibit T-cell cytokine secretion in vitro

To investigate the inhibitory effects that MDSCs exhibit on CD8+ T cells, MDSCs were sorted and co-cultured with CD8+ T cells and tumor cells at the indicated ratios (Fig. 3). Following 48 h, the CD8+ T cells and tumor cells were labeled with Annexin-V-FITC and 7-AAD respectively, followed by detection using flow cytometry. An ELISA was performed to measure IFN-γ levels in the co-culture supernatant. The proportion of CD8+ T-cell apoptosis was significantly increased as the proportion of MDSCs increased (P=0.001; Fig. 3B), whereas the proportion of tumor cell apoptosis significantly decreased (P=0.0017; Fig. 3C). The concentration of IFN-γ significantly decreased with the increase in MDSCs (P=0.0016; Fig. 4), which implies that the MDSCs inhibit T cell cytokine secretion.

 Discussion

The human immune system has evolved over millions of years and can protect the body from pathogens, including bacteria, and parasites (12). Designing an immunotherapy that can enhance the anticancer effects of the immune system remains a challenge and immunotherapies have had little success in clinical trials (13). It has previously been demonstrated that tumor size is not significantly affected following the administration of immunotherapy, which may be due to certain cell types that suppress the immune response (14,15). An optimistic trend in the treatment of lung disease, which may change the immune suppressive effect, is emerging (16). Immunotherapies are designed to stimulate the immune system in order to restore its anticancer effects (17,18). The purpose of the current study was to evaluate whether lung squamous cell carcinoma cells are affected by MDSCs, as there is currently little information on changes in MDSC proportion in patients with tumors. A number of recent studies, performed independently in patients with non-small cell lung cancer (NSCLC) and other carcinomas, demonstrated that these cells are immunosuppressive and that their frequency is upregulated in carcinoma (19–21).

Previous studies have demonstrated the functions of human MDSCs in hepatocellular, renal carcinoma and prostate cancer types, among others (22,23). The proliferation and aggregation of MDSCs in human malignant neoplasms may have an effect on tumor progression and prognosis. The definition of these immunosuppressive cells in patients is problematical, since there is no human homolog of Gr-1 marker, and no correlation between phenotype and immune suppressive properties has been reported (11,24). Identifying MDSCs may aid in developing anticancer treatments that target these cell populations. MDSCs are a heterogeneous group of cells that include monocytic (M)-MDSCs, polymorphonuclear MDSCs and immature myeloid cells. These three subsets can express different combinations of myeloid markers that are associated with different differentiations of myeloid cells (CD14, CD15, HLA-DR, CD33, CD11b, CD15 and CD16), and can possess the immunosuppressive activity of MDSCs (25).

M-MDSCs were the first subtype of human MDSCs to be identified in the peripheral blood of melanoma patients, and are defined as CD14+ and HLA-DR−/low cells (26). M-MDSCs have also been identified in a number of other cancer types, including renal cell carcinoma, hepatocellular carcinoma and advanced NSCLC (27). In the present study, following the exclusion of debris and granulocytes, CD14+ and HLA-DR−/low cells were selected, as these cells have been widely studied. The frequency of these cells has been reported to be elevated in a number of cancer types (28); however, further studies are required in patients with lung squamous cell carcinoma. Data from the present study demonstrated that the frequency of MDSCs in the PBMCs of 78 patients with lung squamous cell carcinoma was significantly increased compared with that of healthy controls. Additionally, the frequency of MDSCs was associated with TNM stage, and the levels of CD4+ and CD8+ T-cells were significantly decreased in patients with lung squamous cell carcinoma compared with healthy controls. Previous studies have demonstrated that the decreased number of lymphocytes described in patients with cancer is partially due to the immunosuppressive effects of MDSCs (29,30).

According to previous studies, MDSCs mediate immunosuppression through a number of molecular mechanisms. MDSCs deplete essential metabolites for T lymphocytes through the activation of arginase-1 and nitric oxide synthase 2 (31). High levels of reactive oxygen species affect T cells by downregulating T-cell surface glycoprotein CD3 ζ chain expression and reducing cytokine secretion. MDSCs interfere with T cell migration and viability by expressing the metalloproteinase disintegrin and metalloproteinase domain-containing protein 17 that is able to cleave the integrin CD62L on T cells. MDSCs promote the clonal expansion of antigen-specific natural regulatory T cells (Tregs) and induce the conversion of CD4+ T cells into induced Tregs through the release of transforming growth factor-β (32). In order to further verify the immunosuppressive effect of MDSCs in lung squamous carcinoma cell immunity, MDSCs from patients with lung squamous cell carcinoma and healthy controls were sorted and subsequently cultured with NCI-H226 cells. As the ratio of MDSCs increased, the proportion of CD8+ T cell apoptosis significantly increased, whereas NICI-H226 cell apoptosis signficantly decreased. Additionally, the concentration of IFN-γ significantly decreased with the increase in MDSCs, which implies that MDSCs inhibit T cell cytokine secretion. This confirms the immunosuppressive effect of MDSCs in lung squamous cell carcinoma (33). These results may aid in developing novel treatments that inhibit malignant neoplasm progression and metastasis. It has previously been reported that it is possible to use MDSCs as a therapeutic target (34).

The present study investigated the proportion of peripheral CD14+HLA-DR−/low MDSCs in patients with different stages of lung squamous cell carcinoma, and the association between different tumor stages and MDSC function. The frequency of MDSCs is significantly increased in patients with lung squamous cell carcinoma. The frequencies of CD4+ T cells and CD8+ T cells in PBMCs from patients with lung squamous cell carcinoma were significantly decreased compared with those from the healthy controls. MDSCs inhibit T-cell cytokine secretion in vitro. In conclusion, MDSCs participate in the immune escape of lung squamous cell carcinoma, and may provide a possible therapeutic strategy for the treatment of this disease.

 References ZhouCLung cancer molecular epidemiology in China: Recent trendsTransl Lung Cancer Res3270279201425806311 LandSRLiuQWickerhamDLCostantinoJPGanzPACigarette smoking, physical activity, and alcohol consumption as predictors of cancer incidence among women at high risk of breast cancer in the NSABP P-1 trialCancer Epidemiol Biomarkers Prev23823832201410.1158/1055-9965.EPI-13-1105-T24569437 ZhouQHFanYGBuHWangYWuNHuangYCWangGWangXYQiaoYLChina national lung cancer screening guideline with low-dose computed tomography (2015 version)Thorac Cancer6812818201510.1111/1759-7714.1228726557925 MotallebnezhadMJadidi-NiaraghFQamsariESBagheriSGharibiTYousefiMThe immunobiology of myeloid-derived suppressor cells in cancerTumour Biol3713871406201610.1007/s13277-015-4477-926611648 QuPWangLZLinPCExpansion and functions of myeloid-derived suppressor cells in the tumor microenvironmentCancer Lett380253256201610.1016/j.canlet.2015.10.02226519756 OchandoJCondePBronteVMonocyte-derived suppressor cells in transplantationCurr Transplant Rep2176183201510.1007/s40472-015-0054-926301174 HaileLAvon WasielewskiRGamrekelashviliJKrügerCBachmannOWestendorfAMBuerJLiblauRMannsMPKorangyFGretenTFMyeloid-derived suppressor cells in inflammatory bowel disease: A new immunoregulatory pathwayGastroenterology135871881881.e1-e5200810.1053/j.gastro.2008.06.03218674538 DetterbeckFCPostmusPETanoueLTThe stage classification of lung cancer: Diagnosis and management of lung cancer, III ed: American College of Chest Physicians evidence-based clinical practice guidelinesChest1435 Supple191Se210S201310.1378/chest.12-235423649438 JettJRSchildSEKeslerKAKalemkerianGPTreatment of small cell lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelinesChest1435 Supple400Se419S201310.1378/chest.12-236323649448 TravisWDBrambillaERielyGJNew pathologic classification of lung cancer: Relevance for clinical practice and clinical trialsJ Clin Oncol319921001201310.1200/JCO.2012.46.927023401443 ZhangGHuangHZhuYYuGGaoXXuYLiuCHouJZhangXA novel subset of B7-H3+CD14+HLA-DR-/low myeloid-derived suppressor cells are associated with progression of human NSCLCOncoimmunology4e977164201510.4161/2162402X.2014.97716425949876 SimonAKHollanderGAMcMichaelAEvolution of the immune system in humans from infancy to old ageProc Biol Sci28220143085201510.1098/rspb.2014.308526702035 AlizadehDLarmonierNChemotherapeutic targeting of cancer-induced immunosuppressive cellsCancer Res7426632668201410.1158/0008-5472.CAN-14-030124778417 KeskinovAAShurinMRMyeloid regulatory cells in tumor spreading and metastasisImmunobiology220236242201510.1016/j.imbio.2014.07.01725178934 LabordeRRLinYGustafsonMPBulurPADietzABCancer vaccines in the world of immune suppressive monocytes (CD14(+)HLA-DR (lo/neg) cells): The gateway to improved responsesFront Immunol5147201410.3389/fimmu.2014.0014724772111 BruchardMGhiringhelliFImpact of chemotherapies on immunosuppression and discovery of new therapeutic targetsBull Cancer1016056072014(In French)24899072 KennedyDEKnightKLInhibition of B lymphopoiesis by adipocytes and IL-1-producing myeloid-derived suppressor cellsJ Immunol19526662674201510.4049/jimmunol.150095726268654 MichaudHAEliaouJFLafontVBonnefoyNGrosLTumor antigen-targeting monoclonal antibody-based immunotherapy: Orchestrating combined strategies for the development of long-term antitumor immunityOncoimmunology3e955684201410.4161/21624011.2014.95568425941618 AlbeituniSHDingCLiuMHuXLuoFKloeckerGBousamraMIIZhangHGYanJYeast-derived particulate beta-glucan treatment subverts the suppression of myeloid-derived suppressor cells (MDSC) by inducing polymorphonuclear MDSC apoptosis and monocytic MDSC differentiation to APC in cancerJ Immunol19621672180201610.4049/jimmunol.160034626810222 KoinisFVetsikaEKAggourakiDSkalidakiEKoutoulakiAGkioulmpasaniMGeorgouliasVKotsakisAEffect of first-line treatment on myeloid-derived suppressor cells' subpopulations in the peripheral blood of patients with non-small cell lung cancerJ Thorac Oncol1112631272201610.1016/j.jtho.2016.04.02627178984 HuangAZhangBWangBZhangFFanKXGuoYJIncreased CD14(+)HLA-DR(−/low) myeloid-derived suppressor cells correlate with extrathoracic metastasis and poor response to chemotherapy in non-small cell lung cancer patientsCancer Immunol Immunother6214391451201310.1007/s00262-013-1450-623760662 UmanskyVSevkoAGebhardtCUtikalJMyeloid-derived suppressor cells in malignant melanomaJ Dtsch Dermatol Ges12102110272014(In English, German)10.1111/ddg.12411_suppl25263083 AlbeituniSHDingCLiuMHuXLuoFKloeckerGBousamraMIIZhangHGYanJYeast-derived particulate β-glucan treatment subverts the suppression of myeloid-derived suppressor cells (MDSC) by inducing polymorphonuclear MDSC apoptosis and monocytic MDSC differentiation to APC in cancerJ Immunol19621672180201610.4049/jimmunol.160034626810222 HuangAZhangBWangBZhangFFanKXGuoYJIncreased CD14(+)HLA-DR(−/low) myeloid-derived suppressor cells correlate with extrathoracic metastasis and poor response to chemotherapy in non-small cell lung cancer patientsCancer Immunol Immunother6214391451201310.1007/s00262-013-1450-623760662 JiangJGuoWLiangXPhenotypes, accumulation, and functions of myeloid-derived suppressor cells and associated treatment strategies in cancer patientsHum Immunol7511281137201410.1016/j.humimm.2014.09.02525305034 GutknechtMFBoutonAHFunctional significance of mononuclear phagocyte populations generated through adult hematopoiesisJ Leukoc Biol96969980201410.1189/jlb.1RI0414-195R25225678 YinYHuangXLynnKDThorpePEPhosphatidylserine-targeting antibody induces M1 macrophage polarization and promotes myeloid-derived suppressor cell differentiationCancer Immunol Res1256268201310.1158/2326-6066.CIR-13-007324777853 Du FourSMaenhoutSKNiclouSPThielemansKNeynsBAertsJLCombined VEGFR and CTLA-4 blockade increases the antigen-presenting function of intratumoral DCs and reduces the suppressive capacity of intratumoral MDSCsAm J Cancer Res625142531201627904768 LiuJZhouYHuangQQiuLCD14+HLA-DRlow/− expression: A novel prognostic factor in chronic lymphocytic leukemiaOncol Lett911671172201525663875 ShiGWangHZhuangXMyeloid-derived suppressor cells enhance the expression of melanoma-associated antigen A4 in a Lewis lung cancer murine modelOncol Lett11809816201626870289 DraghiciuOLubbersJNijmanHWDaemenTMyeloid derived suppressor cells-An overview of combat strategies to increase immunotherapy efficacyOncoimmunology4e954829201510.4161/21624011.2014.95482925949858 Ostrand-RosenbergSMyeloid-derived suppressor cells: More mechanisms for inhibiting antitumor immunityCancer Immunol Immunother5915931600201010.1007/s00262-010-0855-820414655 SolitoSMarigoIPintonLDamuzzoVMandruzzatoSBronteVMyeloid-derived suppressor cell heterogeneity in human cancersAnn N Y Acad Sci13194765201410.1111/nyas.1246924965257 de SanctisFSolitoSUgelSMolonBBronteVMarigoIMDSCs in cancer: Conceiving new prognostic and therapeutic targetsBiochim Biophys Acta18653548201626255541

Flow cytometry analysis to quantify the frequency of MDSCs in patients with lung squamous cell carcinoma. (A) Gating strategy used for the identification of CD14+ HLA-DR−/low MDSCs and representative flow cytometry data. (B) Quantification of the frequency of MDSCs from 78 patients with lung squamous cell carcinoma and 30 healthy controls. MDSCs, myeloid-derived suppressor cells; CD14, monocyte differentiation antigen CD14; HLA-DR, HLA class II histocompatibility antigen DR; HC, healthy control; LC, lung squamous cell carcinoma.

Frequency of MDSCs positively correlates with TNM stage in patients with lung squamous cell carcinoma. (A) Frequency of MDSCs in the peripheral blood of patients with different TNM stages (II, III and IV) of lung squamous cell carcinoma. (B) Frequency of CD4+ cells in healthy controls and lung squamous carcinoma patients. (C) Frequency of CD8+ cells in healthy controls and lung squamous carcinoma patients. MDSCs, myeloid-derived suppressor cells; HC, healthy control; LC, lung squamous cell carcinoma; CD4, T-cell surface glycoprotein CD4; CD8, T-cell surface glycoprotein CD8; TNM, tumor-node-metastasis.

MDSCs inhibit T cell cytokine secretion in vitro. (A) Analysis of NCI-H226 and CD8+ cell apoptosis. (B) Statistical analysis of apoptosis of CD8+ T cells and NCI-H226 cells. (C) Statistical analysis of apoptosis of NCI-H226 cells.

Concentration of IFN-γ in the supernatant of the T-cell surface glycoprotein CD8+ T cell/MDSC co-culture at the indicated ratios. MDSCs, myeloid-derived suppressor cells; IFN-γ, interferon-γ.

Clinicopathological characteristics of patients with lung squamous cell carcinoma and healthy controls.

Clinicopathological characteristics Healthy controls Patients with lung squamous cell carcinoma
Total, n 30 78
Age, years (mean ± SD) 58.4±8.9 63.4±9.2
Gender (male/female) 26/4 68/10
Tumor-node-metastasis stage
  II ND 15
  III ND 37
  IV ND 26
SCC antigen, µg/l (mean ± SD) ND 23.8±1.5
CEA, ng/l (mean ± SD) ND 135.9±34.1

SCC, squamous cell carcinoma-associated; CEA, carcinoembryonic antigen; SD, standard deviation; ND, no data.