Cellular characterization of immune cells in non-small cell lung cancer and mesothelioma

Lung cancer is the leading cause of cancer deaths worldwide for both women and men. Mesothelioma, form of cancer concerning membrane surrounding the lungs, is under-examined especially in terms of immune cells. Tumor microenvironment mainly affects immunological responses via either suppressing effector cells or converting them to the regulatory cell types. Secretion of immune suppressor cytokines by regulatory cell types contributes to suppression of immune responses within cancer microenvironment. Regulatory cell types including regulatory T and B cells contribute to the inhibitory responses with their ability to secrete anti-inflammatory cytokines such as Interleukin-10 (IL-10). This study aims to characterize immune cells in non-small cell lung cancer and mesothelioma according to cell surface molecules and cytokine profiles of the cells. Cellular characterization of effector and regulatory immune cells in blood and tumor tissue samples of non-small cell lung cancer and mesothelioma patients is aimed to be performed. Our second aim was to investigate the effects of NSCLC microenvironment on healthy T and B cells. Overall, this study especially aims to identify types of peripheral and tumor infiltrating regulatory B cells that are not identified before in lung cancer and mesothelioma. Most of the studies in the literature regarding regulatory B cells are restricted to the studies of peripheral blood, which reflects only a systemic immune responses for NSCLC. For that reason, investigating regulatory B cells within its microenviroment is necessary. To investigate the relationship among CD4+ T cells, CD19+ B cells and their subsets, T cell specific (CD4 and CD8), regulatory B cell specific (CD19, CD24 ve CD38), and PD-1 and PD-L1 cell surface molecules were examined along with anti-inflammatory (IL-10) and pro-inflammatory (IFNγ and IL-17) cytokine profiles. In this study, we performed flow cytometry analysis of peripheral blood and tumor tissue samples collected from patients with NSCLC and mesothelioma from Istanbul University, Cerrahpaşa Medical School, Thoracic Surgery Department. Peripheral Mononuclear Blood Cells (PBMC) were isolated from both healthy and patient samples. Tumor infiltrating lymphocytes were obtained from tumor tissues that were resected during surgery. PBMC and TILs were stimulated with CpG ODN K3 (0,7 μg/ml) for 24 hours and PMA/ionomycin before staining of IL-10 and regulatory B cell surface markers. Immune cell characterization was performed by flow cytometry using anti-CD4, anti-CD8, anti-CD19, anti-CD24, anti-CD38, anti-PD1, anti-IL10, anti-IFNγ and anti-IL-17 antibodies. To study the effects of NSCLC microenvironment, CD4+ T cells and B cells were purified from healthy PBMCs by magnetic cell sorting with a column free commercial reagent kit (Stemcell). Then, B and T cells were cultured in either tumor or normal tissue conditioned medium which were obtained by culturing cells of tumor and normal tissue for 2 days. After that, IL10, IFNγ and PD-L1 expression were analyzed. Proliferation assay was also performed for T cells. 7 healty controls (3 females and 4 males, median age was 36 years; range, 25 to 52 years), 16 non-small lung cancer (3 females and 13 males, median age was 59 years; range, 42 to 69) and 3 mesothelioma (3 males, median age was 66 years; range 55 to 78) patients were analyzed. IL-10 expression of CD4+ and CD19+ (0.82-24.1%; 0.85-17.3%) cells were increased in bloods and also in TILs (0-23.7%; 0-20%) of patients with NSCLC compared with healthy individuals. CD4+ IL10+ (0-35.7%) cell frequencies were significantly increased in blood samples of mesothelioma patients. In addition, CD19+IL10+ cells were significantly increased in their tumors. IL-10 expression by CD19+CD24+CD38+ B cells (0-75%) from lung cancer patients' blood were greater than CD19+CD24+CD38+ B cells of healthy controls. PD-1 and IL-10 double positive B cells were significantly increased in PBMCs of LC patients (0,68-5,45%) in comparison to healthy individuals (0-0,94%). We observed increased IL-10 expression by both CD4+ T cells (0.82-24.1%) and CD19+ B cells (0.85-17.3%) for blood samples of the patients with lung cancer. Moreover, there was a high incidence of IL-10 expression in their tumor infiltrating CD4+ T cells (0-23,7%) and CD19+ B cells (0-20%). In addition, tumor infiltrating CD19+IL-10+ B cells (1,15-10%) were significantly increased in mesothelioma patients. Our results were also showed that IFNγ+ cytotoxic T cells were significantly decreased in TIL of lung cancer patients to the healthy control level compared with their blood samples (20,3-94,9% to 3,03-53,2%). In addition, IFNγ producing CD4+ T cell frequencies were significantly decreased in bloods of LC patients (10,4-38,3% to 1,82-21,4%). In turn, increased CD4+IL-10+ T cell frequencies suggests that CD4+ T cell response shift to the anti-inflammatory responses for both systemic and local responses. Moreover, our in vitro studies showed that IFNγ expression by CD4+ T cells decreased (15,2 to 10,7%) in tumor microenvironment compared to normal one. Also, IL-10 production was significantly elevated in healthy control B cells when they were cultured with supernatant of primary NSCLC tumor cells. Even though, we have low numbers of samples, the frequency of CD19+ CD24+ CD38+ IL-10+ B cells (0-75%) were highly increased in for NSCLC. PD-1 expression on B cells were significatly increased in both blood (2,14-11,1%) and tumor tissue (3,52-28,6%) samples of NSCLC patients compared to healthy individuals (0,79-3,9%). Moreover, PD-1 and IL-10 double positive B cells were significantly increased in both blood (0,68-5,45%) and tumor tissue samples (1,58-7,04%) of patients with NSCLC compared to healthy individuals (0-0,94%). Altogether, regulatory B cels were significantly elevated in both blood and TILs of patients with NSCLC while they were significantly elevated in TILs of patients with mesothelioma. We also observed that IL-10 production levels of PD-L1+ B cells in tumor microenviroment were increased. In conclusion, these results indicate an anti-inflammatory environment within tumor region both in lung cancer and mesothelioma.