Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the world. Immunological analysis of the tumor microenvironment (immunoscore) shows great promise for improved ...prognosis and prediction of response to immunotherapy. However, the exact immune cell composition in NSCLC remains unclear. Here, we used flow cytometry to characterize the immune infiltrate in NSCLC tumors, non-cancerous lung tissue, regional lymph node, and blood. The cellular identity of >95% of all CD45
immune cells was determined. Thirteen distinct immune cell types were identified in NSCLC tumors. T cells dominated the lung cancer landscape (on average 47% of all CD45
immune cells). CD4
T cells were the most abundant T cell population (26%), closely followed by CD8
T cells (22%). Double negative CD4
CD8
T cells represented a small fraction (1.4%). CD19
B cells were the second most common immune cell type in NSCLC tumors (16%), and four different B cell sub-populations were identified. Macrophages and natural killer (NK) cells composed 4.7 and 4.5% of the immune cell infiltrate, respectively. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c
DCs, and CD141
DCs) which together represented 2.1% of all immune cells. Among granulocytes, neutrophils were frequent (8.6%) with a high patient-to-patient variability, while mast cells (1.4%), basophils (0.4%), and eosinophils (0.3%) were less common. Across the cohort of patients, only B cells showed a significantly higher representation in NSCLC tumors compared to the distal lung. In contrast, the percentages of macrophages and NK cells were lower in tumors than in non-cancerous lung tissue. Furthermore, the fraction of macrophages with high HLA-DR expression levels was higher in NSCLC tumors relative to distal lung tissue. To make the method readily accessible, antibody panels and flow cytometry gating strategy used to identify the various immune cells are described in detail. This work should represent a useful resource for the immunomonitoring of patients with NSCLC.
Cytokine gene delivery by viral vectors is a promising novel strategy for cancer immunotherapy. Semliki Forest virus (SFV) has many advantages as a delivery vector, including the ability to (i) ...induce p53-independent killing of tumor cells
apoptosis, (ii) elicit a type-I interferon (IFN) response, and (iii) express high levels of the transgene. SFV vectors encoding cytokines such as interleukin (IL)-12 have shown promising therapeutic responses in experimental tumor models. Here, we developed two new recombinant SFV vectors encoding either murine tumor necrosis factor-α (TNF-α) or murine interferon-γ (IFN-γ), two cytokines with documented immunostimulatory and antitumor activity. The SFV vector showed high infection rate and cytotoxicity in mouse and human lung carcinoma cells
. By contrast, mouse and human macrophages were resistant to infection with SFV. The recombinant SFV vectors directly inhibited mouse lung carcinoma cell growth
, while exploiting the cancer cells for production of SFV vector-encoded cytokines. The functionality of SFV vector-derived TNF-α was confirmed through successful induction of cell death in TNF-α-sensitive fibroblasts in a concentration-dependent manner. SFV vector-derived IFN-γ activated macrophages toward a tumoricidal phenotype leading to suppressed Lewis lung carcinoma cell growth
in a concentration-dependent manner. The ability of SFV to provide functional cytokines and infect tumor cells but not macrophages suggests that SFV may be very useful for cancer immunotherapy employing tumor-infiltrating macrophages.
Rituximab is a monoclonal antibody that targets the CD20 B-cell-specific antigen and is widely used as therapy for B-cell lymphoma. Since rituximab depletes both malignant and normal B cells, it is ...increasingly being used to treat various conditions in which normal B cells have a pathogenic role, such as rheumatoid arthritis and multiple sclerosis. It is well-established that rituximab efficiently eliminates B cells in blood, lymph nodes, and spleen. In contrast, the effect of rituximab in non-lymphoid tissues remains poorly documented and is debated. Here, we report a rheumatoid arthritis patient who was treated with rituximab before receiving thoracic surgery for non-small cell lung cancer. Using flow cytometry and immunohistochemistry, we show that rituximab efficiently depleted CD20-positive B cells in a primary lung tumor, in lung-associated lymph nodes, and in normal lung tissue. We conclude that rituximab may be very efficient at depleting normal B cells in the lungs. This property of rituximab may potentially be exploited for the treatment of conditions in which pathogenic B cells reside in the lungs. On the other hand, the clearance of lung B cells may provide an explanation for the rare cases of severe non-infectious pulmonary toxicity of rituximab.
Pulmonary typical carcinoid (TC) is a low‐grade, rare lung cancer of neuroendocrine origin. Currently, there is very little information available about the immune cell composition in TC tumours. ...Here, we analysed by flow cytometry resected tumours from four never‐smoker female patients with TC. Twelve distinct immune cell types were identified in TC tumours. The most abundant immune cells were CD8+ T cells, CD4+ T cells, B cells and macrophages, which represented 19.8%, 17.7%, 11.5% and 11% of all tumour‐infiltrating CD45+ leucocytes, respectively. Natural killer (NK) cells (8.8%) and neutrophils (3.9%) were also common. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c DCs, and CD141 DCs) which together constituted 1.4% of all immune cells in TC tumours. Small populations of basophils (1.2%), mast cells (0.8%) and eosinophils (0.6%) were also present. Notably, the percentage of leucocytes (of all living cells) was much lower in TC tumours compared to high‐grade non‐small cell lung cancer (NSCLC) tumours and also compared to non‐cancerous lung tissue. We conclude that TC tumours are relatively non‐inflammatory, although the immune landscape was found to be very complex.
The analysis of tumour‐associated macrophages (TAMs) has a high potential to predict cancer recurrence and response to immunotherapy. However, the heterogeneity of TAMs poses a challenge for ...quantitative and qualitative measurements. Here, we critically evaluated by immunohistochemistry and flow cytometry two commonly used pan‐macrophage markers (CD14 and CD68) as well as some suggested markers for tumour‐promoting M2 macrophages (CD163, CD204, CD206 and CD209) in human non–small cell lung cancer (NSCLC). Tumour, non‐cancerous lung tissue and blood were investigated. For immunohistochemistry, CD68 was confirmed to be a useful pan‐macrophage marker although careful selection of antibody was found to be critical. The widely used anti‐CD68 antibody clone KP‐1 stains both macrophages and neutrophils, which is problematic for TAM quantification because lung tumours contain many neutrophils. For TAM counting in tumour sections, we recommend combined labelling of CD68 with a cell membrane marker such as CD14, CD163 or CD206. In flow cytometry, the commonly used combination of CD14 and HLA‐DR was found to not be optimal because some TAMs do not express CD14. Instead, combined staining of CD68 and HLA‐DR is preferable to gate all TAMs. Concerning macrophage phenotypic markers, the scavenger receptor CD163 was found to be expressed by a substantial fraction (50%‐86%) of TAMs with a large patient‐to‐patient variation. Approximately 50% of TAMs were positive for CD206. Surprisingly, there was no clear overlap between CD163 and CD206 positivity, and three distinct TAM sub‐populations were identified in NSCLC tumours: CD163+CD206+, CD163+CD206− and CD163−CD206−. This work should help develop macrophage‐based prognostic tools for cancer.
The formation of the complex between N-acetyl-L-cysteine and palladium(II) chloride in Britton-Robinson buffer solution in the pH range 2.08-8.00 was studied. The optimum conditions for this reaction ...were ascertained and a spectrophotometric method was developed for the determination of N-acetyl-L-cysteine in the concentration range 4.0-65.3 micrograms ml-1, using PdCl2 as the reagent. The detection limit was 1.63 micrograms ml-1 and the relative standard deviation varied from 0.63 to 1.92%. The method was applied to the determination of N-acetyl-L-cysteine in water and in injection solutions.
A simple and rapid method for the spectrophotometric determination of pralidoxime chloride in urine, based on complex formation with palladium(II) without preliminary mineralization of the sample, is ...described. The proposed method is shown to be reproducible and in good agreement with a reference method, which involved spectrophotometric determination of corresponding oximate ions in ammonium hydroxide solution at 336 nm. Our results indicate that the proposed method is reliable, rapid and of sufficient sensitivity for pralidoxime chloride analysis in urine.
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