Mediastinal lymph node removal modulates natural killer cell exhaustion in patients with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death globally. In this study, the effect of complete removal of mediastinal lymph nodes by video-assisted mediastinoscopic lymphadenectomy (VAMLA) on natural killer (NK) cell phenotype and functions in patients with NSCLC was evaluated. The study included 21 NSCLC patients (cIA-IVA) undergoing VAMLA staging and 33 healthy controls. Mononuclear cells were isolated from peripheral blood of all participants and mediastinal lymph nodes of the patients. NK cells were analyzed by flow cytometry to define NK subsets, expressions of PD-1, CTLA-4, activating/inhibitory receptors, granzyme A, and CD107a. The plasma levels of soluble PD-1, PDL-1, and CTLA-4 were measured by ELISA. Mediastinal lymph nodes of NSCLC patients had increased ratios of exhausted NK cells, increased expression of PD-1 and IL-10, and impaired cytotoxicity. Mediastinal lymph nodes removal increased CD56dimCD16bright cytotoxic effector phenotype and reduced exhausted NK cells. PD-1+ NK cells were significantly more abundant in patients' blood, and VAMLA significantly reduced their ratio as well. The ratio of IL-10 secreting regulatory NK cells was also reduced after VAMLA. Blood NK cells had increased cytotoxic functions and spontaneous IFN-γ secretion, and these NK cell functions were also recovered by VAMLA. Mediastinal lymph node removal reversed NK cell exhaustion, reduced regulatory NK cells, and improved antitumoral functions of NK cells. Tumor-draining lymph nodes may contribute to tumor evasion from antitumoral immune responses. The role of their removal needs to be further studied both to better understand this mechanism and as a potential immunotherapeutic approach.

Mediastinal lymph node removal ameliorates cytotoxic T-lymphocyte functions in patients with non-small cell lung cancer.

INTRODUCTION: Mediastinal lymph node (MLN) removal by video-assisted mediastinoscopic lymphadenectomy (VAMLA) for preoperative cancer staging was reported to be associated with increased survival. The aim of this study was to evaluate the immunologic effects of complete MLN removal by VAMLA on cytotoxic T lymphocyte (CTL) phenotype and function. METHODS: Seventeen patients with non-small cell lung cancer (NSCLC) (stage cT1-4N0-3M0-1A) and 20 healthy participants were included in this study. Blood samples were collected before and 4 weeks after the procedure. Lymphocytes were isolated from the removed MLNs. CTL phenotypes and functions were evaluated by flow cytometry. Plasma levels of soluble programmed cell death protein 1 (sPD-1), soluble programmed cell death protein 1 ligand, and soluble CTL antigen 4 (sCTLA-4) were measured with enzyme-linked immunosorbent assay. RESULTS: The ratio of the immunosenescent CTLs (CD3+CD8+CD28-) was increased in peripheral blood and MLNs of the patients with NSCLC compared to controls (p = 0.037), and MLN removal did not change this ratio. PD-1 and CTL antigen 4 expressions were significantly reduced in peripheral blood CTLs after MLN removal by VAMLA (p = 0.01 and p = 0.01, respectively). Granzyme A expression was significantly reduced in the peripheral blood CTLs of the patients compared to controls (p = 0.006) and MLN removal by VAMLA significantly improved Granzyme A expression in CTLs (p = 0.003). Plasma concentrations of sPD-1 and sCTLA-4 remained unchanged after VAMLA. CONCLUSION: CTLs in the MLNs and peripheral blood of the patients with NSCLC had an immunosenescent phenotype, increased immune checkpoint receptor expression, and impaired cytotoxicity. MLN removal by VAMLA improved these phenotypic and functional characteristics of CTLs. These changes may explain the potential contribution of VAMLA to improved survival.

Functionality of natural killer cells in obese asthma phenotypes.

BACKGROUND: Obesity-associated asthma (OA) is a difficult to treat asthma phenotype due to its severity and poor response to inhaled steroids. Early-onset allergic (EoOA) and late-onset non-allergic (LoOA) OA are suggested subtypes of this phenotype. Natural Killer (NK) cells are key elements of innate immunity involved in cytotoxicity and immune regulation, with uncertain role in OA pathogenesis. METHODS: Early-onset allergic and LoOA patients together with obese non-asthmatic (ONA) controls have been enrolled in the study. Peripheral blood samples have been collected for analysis. Percentages of total NK cells, CD3- CD56dim and CD3- CD56bright NK cell subsets, cytotoxic activity, intracellular interferon-γ, interleukin (IL)-10, IL-13, IL-17 secretion and activatory receptors (NKG2D, NKp46i and NKp44) have been investigated by flow cytometry. The effect of IL-12 and IL-23 stimulation on NK cells and intracellular cytokines in different groups have also been analysed and compared with unstimulated conditions. RESULTS: Results of ONA (n = 5, age 42 ± 8), EoOA (n = 5, age 42 ± 10) and LoOA (n = 8, age 46 ± 8) patients have analysed. Body Mass Index has been found to be negatively correlated with CD69 (p = .022, r = -0.534). NKG2D receptor has been significantly low in CD56dim cells of asthma population (p = .046). NKp44 receptor expression has increased after IL-12 stimulation in EoOA and control group (p = .02). Intracellular IL-10 content has increased in LoOA and control subjects (p = .018, p = .03) but not in the EoOA group. Intracellular IL-17 level has found be higher in allergic OA group. LoOA patients showed a decreased NK cytotoxicity compared with the early-onset asthma group (p = .05). CONCLUSION: Our study suggests an impaired NK receptor expression, activation and reduced cytotoxicity in OA patients together with variances between different subtypes of this phenotype. This data would be beneficial for tailoring a more personalized treatment strategy combatting steroid resistance and frequent exacerbations in this group of patients.

PD-1, CTLA-4, LAG-3, and TIGIT: The roles of immune checkpoint receptors on the regulation of human NK cell phenotype and functions.

The roles of immune checkpoint receptors were defined in many cancers and autoimmune diseases, while there is limited information on their functional roles in the NK cells of healthy individuals. Immune checkpoint receptor expression of NK cell subsets and their association with NK cell functions (cytotoxic capacity and cytokine production) in healthy population were investigated. PD-1, CTLA-4, LAG-3 and TIGIT expression of peripheral blood NK cells, cytokine levels (TNF-α, IFN-γ, IL-10) and cytotoxic functions (granzyme A, perforin, CD107a; with/without K562 target cell stimulation) were evaluated by flow cytometry. CD56dimCD16dim NK cells had the highest expression of TIGIT, while CD56dimCD16- NK cells had highest expression of PD-1, CTLA-4 and LAG-3. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had increased amount of IL-10 however, reduced IFN-γ and TNF-α levels. Cytotoxic granule expressions (perforin and granzyme A) were reduced in PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells. However, TIGIT expression did not alter perforin and granzyme A expressions. Degranulation capacity was reduced in three groups of NK cells (PD-1+ or LAG-3+ or TIGIT+). TIGIT+ NK cells responded strongly to target cell stimulation, while NK cells in the other groups (PD-1+ or CTLA-4+ or LAG-3+) were resistant. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had a regulatory phenotype, impaired cytotoxic functions, and response to target cell stimulation. In contrast, TIGIT+ NK cells had strong baseline cytotoxic activity that further increased in response to target cell stimulation.

The role of atopy in the pathogenesis of bleomycin pulmonary toxicity.

INTRODUCTION: Bleomycin pulmonary toxicity (BPT) is a potentially life-threatening consequence of bleomycin usage in patients. An overproduction of epithelium-derived cytokines, habitually linked to allergic inflammation, has been recently revealed in experimental models of BPT. METHODS: We assessed retrospectively our cohort of patients with Hodgkin Lymphoma treated with bleomycin between 2014 and 2016 for their demographic, clinical features, including BPT development, atopy status and risk factors for BPT. Then they were invited for allergy testing and blood sample collection. The samples were stimulated with different stimuli (Bleomycin, IL-33, TSLP) for 24 h on cell culture. The culture supernatants were analysed for TGF-ß, Galectin3, Arginin, Amphiregulin, Eotaxin, IFNγ, TNFα, IL1ß, 4, 5, 6, 10, 13, 17, MIP-1α, and bleomycin hydrolase (BLH) levels. RESULTS: The cohort consisted of 51 patients showed that atopy was the only significant risk factor for BPT occurrence (OR: 7.2, p = 0.007). Fourteen subjects were included for blood analysis. The analysis of supernatants at the unstimulated condition revealed that BLH and Amphiregulin were significantly lower in patients who had BPT than controls. The BLH cut-off that best identified a history of BPT was 175.31 (Sensitivity: 62.5%, specificity: 100%). Following the stimulation, BLH reduced compared to the unstimulated condition and the difference between groups remained significant (p < 0.05). CONCLUSION: Our study is the first to report that low levels of bleomycin hydrolase in allergic individuals may be predisposing to a possible pathway of fibrosis.

Interleukin-10 is increased in successful drug desensitization regardless of the hypersensitivity reaction type.

BACKGROUND: Little is known about the mechanism of desensitization in hypersensitivity drug reactions. OBJECTIVE: The aim of this study was to evaluate the effects of drug desensitization on some cytokine levels in patients desensitized for drug hypersensitivity reactions. METHODS: Patients with a hypersensitivity reaction to any drug for whom desensitization was planned with the culprit drug, patients who could tolerate the same drugs and healthy subjects who were not exposed to these drugs were enrolled. Bead-based Milliplex MAP multiplex technology was used to determine interleukin (IL)-4, IL-5, interferon-γ and IL-10 levels in the sera of the subjects as a baseline and 24 hours after desensitization had been completed in the patients. RESULTS: A total of 26 patients (16 female [61.5%]; mean age 48.46 ± 15.97 years old), 10 control patients (5 female [50%]; mean age 47.4 ± 15.4 years old) and 5 healthy subjects (3 female [60%]; mean age 34.2 ± 5.6 years old) were enrolled. Four of the 26 patients did not tolerate the procedure and were grouped as the 'unsuccessful desensitization group' whereas 22 patients successfully completed the procedure and formed the 'successful desensitization group.' Baseline cytokine levels in the 3 groups were not statistically different. Postdesensitization IL-10 levels in the successful desensitization group were significantly higher than their initial levels (p = 0.005) whereas none of the cytokine levels significantly changed in the unsuccessful desensitization group. The rise in IL-10 levels was greater in chemotherapeutic desensitizations when compared to other drugs (p = 0.006). CONCLUSION: Successful desensitization independent of the hypersensitivity reaction type seems to be related to the increase of IL-10.

Intracellular cytokine and cathelicidin secretion from monocytes and neutrophils in childhood tuberculosis.

Human cathelicidin (hCAP 18) is one of the main immunomodulators in the local immune response to Mycobacterium tuberculosis. There has been no information regarding the role of hCAP 18+ in the in vivo production of cytokine in childhood pulmonary tuberculosis (TB). We aimed to determine the intracellular cytokine secretion, including hCAP 18+ from monocytes and neutrophils of pulmonary TB in children, compared with healthy children. Fifteen patients with pulmonary TB were enrolled in the study as the study group, and 15 healthy children as the control group, between the age of 1 and 16 years. The patients' 25 hydroxyvitamin D levels were measured. The expression of hCAP 18+, TNF α, INF-γ and IL-8 from CD14+ monocytes and CD15 + neutrophils was analyzed using the flow cytometry method. The statistical analysis was performed with PASW Statistics v.13.0. The mean vitamin D level was similar in both groups (P = 0.78). The expression of hCAP 18+ (P = 0.0001) and IL-8 from CD14 + (P = 0.0001) monocytes were significantly higher in the study group compared with the control group. There was no difference in the hCAP 18+ and IL-8 expression in CD 15+ neutrophils in both groups. The expression of TNF α, INF-γ from CD14+ monocytes and CD 15+ neutrophils, in both study and control groups revealed no statistical differences.The level of hCAP 18+ and IL-8 released from monocytes were enhanced in the serum in childhood pulmonary TB. The present study is the first report detecting the intracellular hCAP 18+ expression in vivo in childhood pulmonary TB.