PD-L1+ lung cancer stem cells modify the metastatic lymph-node immunomicroenvironment in nsclc patients.

INTRODUCTION: Cancer stem cells (CSCs) are implicated in tumor initiation and development of metastasis. However, whether CSCs also affect the immune system is not fully understood. We investigated correlations between the PD-L1+ CSCs, changes in T-cell phenotype in metastatic and non-metastatic lymph nodes (LNs) and response to treatment. METHODS: LNs' aspirates were obtained during the EBUS/TBNA procedure of 20 NSCLC patients at different stages of the disease. CSCs and T-cell characteristics were determined by flow cytometry. RESULTS: PD-L1+ CSCs positively correlated with the percentage of Tregs, PD-1+ CD4 T cells and Tim3+ CD4+ T cells, whereas PD-L1+ CSCs were negatively correlated with CD4+ T cells and CD28+ CD4+ T cells. The percentage of PD-L1+ CSCs was higher in patients with progressive disease (PD) as compared to patients with stable disease (SD) or partial response (PR). Among T cells, only PD-1+ CD4+ T cells and Tim3+ CD4+ T-cell frequencies were higher in patients with PD as compared to patients with SD or PR. CONCLUSION: The frequency of PD-L1+ CSCs associates with an altered T-cell frequency and phenotype indicating that CSCs can affect the immune system. The higher percentage of PD-L1+ CSCs in patients with PD may confirm their resistance to conventional therapy, suggesting that CSCs may be an interesting target for immunotherapy.

Causes of Chronic Cough in Non-smoking Patients.

Chronic cough is a common medical problem. The aim of the study was to analyze chronic cough causes in non-smoking patients and to search for demographic factors associated with different cough reasons. The etiology of cough was determined by medical history, diagnostic tests and response to specific treatment. Patients with significant abnormalities in the chest radiograph or spirometry were not included. The study included 131 non-smoking patients; median age 54 years, 77 % female. The most frequent causes of cough were gastroesophageal reflux disease (GERD) (62 %) and upper airway cough syndrome (UACS) (46 %). Cough variant asthma and non-asthmatic eosinophilic bronchitis (NAEB) were diagnosed in 32 (25 %) and 19 (15 %) patients, respectively. Other cough causes were found in 27 patients (21 %). Asthma was a significantly more common cause of chronic cough in women than in men (31 % vs. 3 %, p = 0.005). A reverse relationship was demonstrated for UACS (39 % vs. 67 %, p = 0.01). Patients with chronic cough aged >50 yrs were more likely to be diagnosed with less common cough causes. In conclusion, the most common chronic cough reasons are GERD and UACS. Asthma-related cough is diagnosed more frequently in females, while UACS-related cough is more frequent in males.

CD4+/CD25+ cells in systemic inflammation in COPD.

The autoimmune reaction is recently suspected to play a role in the pathogenesis of chronic obstructive lung disease (COPD). As COPD is a systemic disease, the elements of an autoimmune response in circulatory system is of interest. It has been shown that regulatory T cells are important in the control of autoimmunity. There are some data on a role of adiponectin in the regulation of immune reactions. The objective of this study was to assess the elements of autoimmune reaction in the peripheral blood (PB) of patients with COPD. Twenty-eight patients with mild/moderate COPD and 20 healthy volunteers were investigated. Flow cytometry method with mixtures of monoclonal antibodies anti: CD14/CD45, CD3/CD19, CD4/CD25/CTLA4 and CD8/CD25 were used. Concentration of adiponectin was measured using ELISA method. We observed significantly lower proportion of CD4+/CD25+ as well as CD4+/CD25+ (high) cells in COPD patients than in healthy controls (15.3 versus 17.8% and 0.79 versus 1.54%, respectively, P < 0.05). The proportion of CTLA4+ cells in CD25+ cells and the mean fluorescence of CTLA4 on CD4+ cells were higher in patients than in healthy controls (10.4 versus 4.7%, P < 0.05, 189% versus 149%, non significant, respectively). We found significantly elevated concentration of adiponectin in patients when compared to healthy subjects (15.4 versus 8.5 µl/ml, P < 0.05). We found that the adiponectin/BMI ratio correlated with the decrease of FEV(1) %. The results of this study support the possible role of CD4/CD25/CTLA4 cells and adiponectin in the systemic inflammation in COPD.

Relationship between airway inflammation and remodeling in patients with asthma and chronic obstructive pulmonary disease.

UNLABELLED: Despite a number of important differences in the pathogenesis, course and prognosis of asthma and chronic obstructive pulmonary disease (COPD), these two entities also have common features with airway inflammation being one of them. Airway remodeling is a characteristic feature of asthma, but data on the bronchial wall thickening in COPD patients are still scarce. AIM: To assess the relation between the inflammatory cell count in the bronchoalveolar lavage fluid (BALF) and thickness of bronchial walls assessed by high resolution computed tomography (HRCT) in asthma and COPD patients. MATERIAL AND METHODS: The study was conducted in 9 patients with mild-to-moderate asthma (M/F 4/5, mean age 35 +/- 10 years) and 11 patients with mild-to-moderate COPD (M/F 7/4, mean age 57 +/- 9 years). In all subjects lung function tests and HRCT scanning of the chest were performed. External (D) and internal (L) diameters of the airways were assessed at five selected lung levels. The lumen area (A(L)), wall area (WA), wall thickness (WT) and bronchial wall thickness (WT/D ratio) were calculated. Eight patients with asthma and 8 patients with COPD underwent fiberoptic bronchoscopy and bronchoalveolar lavage (BAL). Total and differential cell counts were assessed in the BAL fluid. RESULTS: Mean FEV(1)% pred was 80 +/- 19%, and 73 +/- 20% in asthma and COPD patients, respectively (NS). No significant differences in the total and differential cell counts in BALF were found in patients with asthma and COPD. There were no significant differences in the airway diameter or airway wall thickness. The mean inner airway diameter was 1.4 +/- 0.3 and 1.2 +/- 0.3 mm and the mean lumen area was 1.8 +/- 0.7 and 1.6 +/- 0.7 mm(2) in asthma and COPD, respectively (NS). Negative correlations between the eosinophil count in BALF and inner airway diameter (r=-0.7, P<0.05) and lumen area (r=-0.7, P<0.05) were found in asthmatics. There was no significant relationship between the BALF cell count and airway wall thickness in COPD patients. - CONCLUSIONS: In mild-to-moderate asthma and COPD the airway diameter and thickness are similar. In asthmatics, the airway diameter might be associated with eosinophil count in BAL fluid.

Effects of cigarette smoke on the lung and systemic immunity.

The influence of tobacco smoke on human health is still an important problem worldwide. Complex inflammatory processes and changes in the immune system are crucial in the pathogenesis of smoking related disorders like chronic obstructive lung disease (COPD), lung cancer, atherosclerosis. The objective of this review is to present the alterations in the immune system in smokers. The main affected system by cigarette smoke (CS) is the respiratory tract. In bronchial epithelium metaplastic and dysplastic changes are accompanied by elevated expression of adhesion molecules and secretion of many cytokines capable of stimulation immune cells influx. In the population of pulmonary macrophages an elevated proportion of cells, changes in expression surface markers with impaired phagocytic and antigen presenting function are observed. Chronic exposure to CS causes increased production of metalloproteinases (MMP) by macrophages and proteolitic enzymes by neutrophils. These enzymes cause destruction of alveolar wall. Increased apoptosis of lung tissue results in augmentation of foreign material which may play a role of autoantigen and which is a target for cytotoxic/suppressor cells. The role of regulatory T (Treg) cells in this process is recently postulated. Smoking cessation is the most effective method of prophylaxis and treatment of diseases related to tobacco smoking. However many immunological changes in smokers are not completely reversible after quitting smoking.

Role of bronchoalveolar lavage in the initial diagnosis of smoking-related interstitial lung diseases.

Some of interstitial lung diseases have a potential relation to tobacco smoking and are known as smoking-related interstitial lung diseases (sr-ILD). The aim of this study was to present the typical features in bronchoalveolar lavage fluid (BALF) of patients with sr-ILD with special account to the morphology of alveolar macrophages. We have found typical features of sr-LD in 18 patients with interstitial lung disease. All patients were smokers. Pulmonary function was moderately impaired. Control group consisted of 12 asymptomatic smokers and 18 healthy nonsmokers. The median total cell count in BALF of patients with desquamative interstitial pneumonia was 64.5 x 10(6) and was much higher than in asymptomatic smokers--9.0 x 10(6), and healthy nonsmokers--3.25 x 10(6). A high proportion (mean 90%) and number (55.5 x 10(6)) of macrophages with the predominance of pigmented cells was found. Mean proportion of CD14 positive macrophages was 6%, and of CD54 positive macrophages - 62%. We concluded that features in BALF typical for sr-ILD are uncommon among routinely evaluated specimens, but differ markedly from other forms of ILD.

Eosinophilic airway inflammation in chronic obstructive pulmonary disease and asthma.

Eosinophilic airway inflammation is regarded as a typical feature of asthma, while in chronic obstructive pulmonary disease (COPD) neutrophils seem predominant inflammatory airway cells. The aim of the present study was to compare the cellular components of airway inflammation in patients with newly diagnosed mild or moderate COPD and asthma. Seventeen patients with COPD (M/F 10/7, aged 57 +/-11 yr) and 22 patients with asthma (M/F 12/10, aged 36 +/-14 yr) were enrolled into the study. None of the patients has been treated with steroids for at least 3 months. All patients underwent clinical examination, laboratory examinations, skin-prick tests, pulmonary function tests, methacholine challenge test, and sputum induction with the total and differential cell count assessments. We found increased number of eosinophils in both study groups. However, there were no significant differences in the cellular composition of induced sputum between the asthma and COPD patients. We conclude that eosinophils are important inflammatory cells not only in asthma, but also in COPD.

Comparison of cellular and biochemical markers of airway inflammation in patients with mild-to-moderate asthma and chronic obstructive pulmonary disease: an induced sputum and bronchoalveolar lavage fluid study.

Although the clinical pictures of asthma and chronic obstructive pulmonary disease (COPD) may be similar, the pathogenesis differs in many aspects. The aim of the present study was to compare the cellular and biochemical features of airway inflammation in patients with asthma and COPD. The study was conducted in 22 patients with asthma (M/F 12/10, mean age 36 +/-14 years) and 17 patients with COPD (M/F 10/7, mean age 57 +/-11 years). Each patient underwent sputum induction followed by bronchoscopy, and bronchoalveolar lavage. Total and differential cell counts and the concentration of interleukin-8 (IL-8) and myeloperoxidase (MPO) were measured in induced sputum (IS) and BALF. We found no significant differences in the total and differential cell counts in IS between asthma and COPD patients. However, COPD patients showed an increased total macrophage count in BALF compared with asthma patients. The relative eosinophil count in BALF was significantly higher in patients with asthma vs. COPD. The concentration of IL-8 in IS and BALF was significantly higher in patients with COPD vs. asthma patients. The BALF concentration of MPO was significantly higher in patients with COPD compared with asthma patients. We conclude that the comparison of cellular composition and the concentration of inflammatory mediators in IS does not differentiate between asthma and COPD. The evaluation of BALF reveals more differences in the cellular and biochemical features of airways inflammation in patients with asthma and COPD than that of IS.

Does the established cause of chronic cough depend on diagnostic approach?

The objective of the present study was to evaluate the prevalence of chronic cough causes and to compare the efficacy of two diagnostic approaches used in ambulatory vs. hospitalized setting. Eighty patients with chronic cough, 40 in each group, were enrolled into the study. The etiology of cough was determined on medical history and on either basic (in out-patients) or detailed (in in-patients) investigations on most common causes of chronic cough. We diagnosed etiology of cough in all subjects. The most frequent causes of cough were gastroesophageal reflux disease (GERD) and upper airway cough syndrome (UACS). Nonasthmatic eosinophilic bronchitis (NAEB) and multiple cough causes were recognized more often in hospitalized patients (P<0.05). We conclude that the main causes of chronic cough were GERD and UACS. An extensive diagnostic approach allows recognizing NEAB more frequently and reveals the complex nature of chronic cough.

Expression of macrophage surface markers in induced sputum of patients with chronic obstructive pulmonary disease.

The aim of our study was to evaluate cellular content in induced sputum in chronic obstructive pulmonary disease (COPD) in relation to the degree of airway obstruction, macrophage count, and phenotype. We compared the proportion of macrophages and cells expressing the following markers: CD11b, CD14, CD54, and CD71 in induced sputum obtained from patients with mild-to-moderate and severe COPD (n=29)], asymptomatic smokers (n=18), and nonsmokers (n=18). The differential cell count and macrophage phenotypes were examined in induced sputum by immunocytochemistry. We observed a greater proportion of neutrophils and eosinophils and an elevated macrophage count in patients with COPD and in smokers in comparison with nonsmokers. Macrophages in patients with severe airway obstruction were characterized by a significantly elevated expression of CD11b and CD14 markers. There were higher proportions of macrophages with expression of CD11b, CD14, CD54, and CD71 in induced sputum of smokers in comparison with nonsmokers. We concluded that macrophages are the cells involved in the inflammatory process caused by smoking in COPD. The macrophage phenotype with elevated CD11b and CD14 expressions was associated with severe airflow limitation.

Effect of IFN-gamma stimulation on expression of intercellular adhesion molecule-1 (ICAM-1) on alveolar macrophages in patients with non-small cell lung cancer.

An impairment of in vitro cytotoxicity and tumoricidal function of alveolar macrophages (AMs) in patients with lung cancer was reported in a number of studies. The aim of our study was to evaluate the expression of intercellular adhesion molecule-1 (ICAM-1) on AMs after stimulation with interferon-gamma (IFN-gamma) in patients with non-small cell lung cancer (NSCLC). The study was performed in 13 patients with NSCLC, 6 patients with various nonmalignant pulmonary diseases, and 6 healthy volunteers. AMs were isolated from bronchoalveolar lavage fluid (BALF) by adherence and then cultured with or without IFN-gamma for 24 h. The expression of ICAM-1 on AMs was analyzed by flow cytometry. Stimulation with IFN-gamma caused increased expression of ICAM-1 on AMs in all studied groups (p < 0.05). The degree of the increase in ICAM-1 expression on AMs after IFN-gamma stimulation was significantly lower in patients with NSCLC compared with healthy volunteers (p = 0.002) or the other patients (p = 0.022). The results suggest impaired reactivity of ICAM-1 expression on AMs after stimulation with IFN-gamma in patients with NSCLC, which might be involved in functional defects of AMs in patients with NSCLC.

Reactivity of alveolar macrophages in lung cancer patients and healthy subjects: surface ICAM-1 after INF-gamma stimulation in vitro.

The linearity of ICAM- I expression on alveolar macrophages (AM) before and after INF-gamma stimulation in healthy and lung cancer subjects were compared. AM were collected by bronchoalveolar lavage and incubated with/without INF-gamma according to standard procedures. The harvested cells were analyzed by flow cytometry using monoclonal antibodies against leucocytes and macrophages. Only viable cells were analyzed. Stimulation with INF-gamma revealed two AM subpopulations of similar size differentiated in the intensity of ICAM-1 expression. They were not distinctly marked in every studied case. Our preliminary results did not confirm the previously reported decreasing reactivity of AMs after INF-gamma stimulation in lung cancer patients.

T-cell subtypes in bronchoalveolar lavage fluid and in peripheral blood from patients with primary lung cancer.

The changes in local immunology play an important role in lung cancer development. We used bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) for the analysis of cell profiles in patients with primary lung cancer. Twenty-one patients with confirmed primary lung cancer and 13 healthy volunteers were investigated. All persons were smokers. The analysis of T-cell subsets was performed with a flow cytometry method and with the following antibodies: anti CD3, CD4, CD8, CD16, CD25, CD45, CD56, and HLA-DR. We found differences in the proportion of lymphocytes between BALF and PB, and a higher proportion of T cells and a lower proportion of B and natural-killer (NK) cells in BALF. There was a significant difference in the proportion of T-cytotoxic/suppressor lymphocytes, which was elevated in the BALF of patients and decreased in patients' PB. The T-helper:T-cytotoxic/suppressor (Th:Tc/s) ratio was significantly lower in the BALF of patients. These changes were visible in patients with a small cell type. The percentage of T cells with the alpha chain of receptor to IL-2 (IL -R) was lower in the BALF of patients than in the control group. Our observations reflect local changes in lung environment in patients affected with lung cancer.

Expression of Fas antigen in the cells from bronchoalveolar lavage fluid (BALF).

Fas antigen is a cell surface receptor protein that mediates apoptosis expressed in various cells. In this study Fas expression was examined in cells of patients with lung diseases in which changes in the lung immunology were documented. We have performed bronchoalveolar lavage (BAL) in 24 patients with sarcoidosis (8), lung fibrosis (9), primary lung cancer (7), and we compared expression of Fas in BALF cells from all groups and healthy volunteers (6). Fas protein was detected by immunocytochemistry using APAAP technique with an LSAB 2 kit (Dako). Positive reactions for Fas were found in the cytoplasm of epithelial cells, macrophages, neutrophils and lymphocytes (according to the intensity). There were some differences in proportion of positive cells and intensity of reaction between patients with interstitial lung diseases, healthy volunteers as well as patients with lung cancer. Higher expression of Fas in alveolar macrophages was observed in patients with sarcoidosis, lower in patients with lung cancer, lung fibrosis and the lowest in healthy persons. The analysis of Fas antigen expression in the BALF cells may be useful in evaluation of the role of apoptosis in lung homeostasis and pathology.

Flow cytometric evaluation of lymphocyte subpopulations in BALF of healthy smokers and nonsmokers.

The purpose of this work was to evaluate the normal lymphocyte phenotype in the bronchoalveolar lavage fluid (BALF). BAL was carried out in 12 untreated healthy nonsmoking volunteers and in 9 cigarette smokers. For the analysis of lymphocyte subsets by two-color flow cytometry, the monoclonal antibodies used were directed anti: CD3, CD4, CD8, CD16, CD19, D25, CD45, CD56 and anti HLA-DR. An increase in the total number of cells in BALF of smoking persons and increased proportion of macrophages was observed. The percentage of CD8+ lymphocytes was 1.7 times higher, whereas the proportions of CD4+ cells, and a CD4+/CD8+ ratio were lower 1.5 and 2.6 times, respectively, in the BALF of cigarette smoking persons when compared with nonsmoking volunteers. The changes did not depend on the age of the person. In conclusion, we suggest that the decreased CD4/CD8 ratio and the elevated CD8 T cell subset may be regarded as a potential risk factor associated with clinically asymptomatic lung cancer. Moreover, in the interpretation of BALF from patients with pulmonary diseases cell proportions of nonsmoking and of smoking persons should be compared with the respective controls.

Interstitial lung disease in systemic sclerosis: comparison of BALF lymphocyte phenotype and DLCO impairment.

Patients with scleroderma (systemic sclerosis-SSc) frequently develop an interstitial lung disease. The role of lymphocytes in fibrosing alveolitis preceding lung fibrosis has been established. The purpose of this work was to evaluate cell profiles and lymphocyte phenotypes in the bronchoalveolar lavage (BAL) fluid and to correlate them with depression in lung function tests detected by depletion of diffusing capacity (DLCO). BAL was carried out in 25 untreated, non-smoking patients suffering from diffuse scleroderma and in 12 healthy non-smoking volunteers. For the analysis of lymphocyte sub-sets flow cytometry and monoclonal antibodies were used. The following cell sub-types were counted: T lymphocytes, B lymphocytes, helper lymphocytes, suppressor/cytotoxic lymphocytes, natural killer cells, cytotoxic T lymphocytes and activated T lymphocytes. The total cell count was higher in the group of patients with mild and moderate impairment in DLCO. The percentage of lymphocytes was greater in patients with DLCO lower than 65% of the predicted value since neutrophilia was found in patients with severe DLCO depletion, i.e. significant when compared with healthy subjects. The proportions of suppressor/cytotoxic lymphocytes and of activated T lymphocytes were higher in patients than in controls. The statistical analysis revealed significant differences between patients with moderate and mild changes in DLCO and the healthy volunteers. A decreased helper/suppressor ratio was noticed in these patients. We concluded that the BALF lymphocyte phenotype analysis may reflect the features of alveolitis in patients with SSc.

Lymphocyte phenotyping in systemic sclerosis: a flow cytometry analysis of lymphocytes in bronchoalveolar lavage fluid.

OBJECTIVE: Patients with scleroderma (systemic sclerosis [SSc]) frequently develop interstitial lung disease. The purpose of this work was to evaluate the cell profile in the bronchoalveolar lavage fluid (BALF) in patients suffering from a diffuse form of systemic sclerosis as compared with healthy controls. STUDY DESIGN: Bronchoalveolar lavage was carried out in the right middle lobe of 25 untreated, nonsmoking patients with SSc and 12 healthy, nonsmoking volunteers. For the analysis of lymphocyte subsets, the following monoclonal antibodies were used: anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD45, anti-CD56. Also, anti-HLA-DR and flow cytometry were used. RESULTS: We found an increase in the total number of cells with an increase in the percentage of lymphocytes and neutrophils in BALF from patients when compared with controls (P < .05). The proportion of lymphocytes, cytotoxic/suppressor CD8+ and activated lymphocytes T CD25+ were higher in patients' BALF (P < .05). The CD4+/CD8+ ratio in BALF from subjects was significantly lower than in controls. These findings were characteristic of patients with early-stage disease. CONCLUSION: Analysis of the BALF lymphocyte phenotype may be useful in the early detection of lung involvement in patients with SSc.

[Phenotype of lymphocytes in bronchoalveolar lavage fluid of patients with lung fibrosis in the course of scleroderma]. / Fenotyp limfocytów w popluczynach oskrzelowo-pecherzykowych u chorych z wlóknieniem pluc w przebiegu twardziny ukladowej.

Patients with systemic sclerosis frequently develop interstitial lung disease. One of the major sites of pathogenic activity are lymphocytes from lung interstitium. The purpose of this work was phenotype analysis of lymphocytes in bronchoalveolar lavage fluid (BALF) with flow cytofluorometry. We found an increase number of CD8+ lymphocytes and CD8+/T ratio, and an reduced CD4+/CD8+ ratio in patients when compared to healthy volunteers. The diffusing capacity (DLCO) correlated positive with percentage of neutrophils and HLA-DR+ lymphocytes. There are the features of mixed type alveolitis in patients with short disease duration. We found increase number of lymphocytes, neutrophils, CD8+ lymphocytes, HLA-DR+ lymphocytes and CD25+ lymphocytes in this patients. We concluded that fluorocytometric BALF analysis may be useful for the diagnosis and management of lung involvement in patients with scleroderma before the treatment.