p21 and p15 are elevated in IT patients and induce the arrest of the NK cell cycle.

<p>(A) NK cells were stimulated for 30 min, 12 h and 72 h with TGF-β1. Total cell lysates were analysed using immunoblotting with antibodies against Smad-2, Smad-2P, p15 and p21. Data are representative of three independent experiments. (B) NK cells were stimulated for 72 h with IT serum or hepatitis ascites in the absence (−) or presence (+) of TGF-β1 Ab. NK cells were lysed, and the lysates were immunoblotted with Abs against Smad-2, Smad-2P, p15 or p21. Data are representative of three independent experiments. (C) Freshly isolated NK cells were obtained from the peripheral blood of healthy controls and IT-phase patients and analysed by western blotting. The results for Smad-2, Smad-2P, p15 and p21 are shown. Data are representative of at least four independent experiments.</p

Ca²⁺ flux down-regulation is induced by synergy between NKG2D and 2B4 in NK cells from IT patients.

<p>Freshly isolated, resting NK cells from the peripheral blood of HC (A), IA (B) and IT (C) patients were loaded with Fluo-4 and Fura Red, and preincubated with mAbs specific for NKG2D (blue), 2B4 (purple) or both (red) on ice for 30 min. Cells were washed, resuspended in cold HBSS with 1% FBS, and prewarmed at 37°C. Fluorescence was measured by Zeiss 510 confocal microscopy. Sixty seconds after the beginning of each scan, secondary F(ab')² goat anti–mouse IgG or KCL (green) was added to each chamber. Traces of the Fluo-4/Fura Red ratios of the representative NK cells are shown. Fluo-4/Fura Red ratios are plotted as a function of time. Green lines show activation with the isotype control (KCL). Blue and purple lines show activation by the single receptors. Red lines show activation by the combination of both receptors. The experiment shown is representative of five independent experiments. The Ca²⁺ mobilisation induced by NKG2D and 2B4 synergy was measured in >100 NK cells from representative healthy controls or patients at 200 (D) and 500 s (E).</p

CD11b⁻CD27⁻ NK Cells Are Associated with the Progression of Lung Carcinoma

<div><p>NK cells are a major component of the antitumour immune response that limits tumour progression. However, it has been reported that tumour-infiltrating NK (TINK) cells from patients with non-small-cell lung carcinoma (NSCLC) exhibit profound defects in degranulation and IFN-γ production. In support of this notion, we report a novel mechanism associated with tumour escape from NK cell-mediated antitumour immunity in lung carcinoma. In this study, we investigated the phenotypic profile of TINK cells based on the expression of the NK-cell maturation markers CD11b and CD27. Interestingly, we found a substantial CD11b⁻CD27⁻ (DN) NK-cell population harboured within the tumour tissues. The presence of this CD11b⁻CD27⁻ NK subset indicated that the TINK cells were of an immature and inactive phenotype. Remarkably, we determined that the presence of DN NK cells had an impact on the clinical outcomes of patients with NSCLC, as the frequency of tumour-infiltrating DN NK cells was positively correlated with the tumour stage and tumour size. We further used a murine Lewis lung cancer (LLC) model to confirm the correlation between the frequency of tumour-infiltrating DN NK cells and the progression of lung carcinoma. Together, our findings demonstrate that the tumour microenvironment may render TINK cells less tumouricidal and thereby contribute to cancer progression.</p></div

TINK cells display an immature phenotype.

<p>(A) Representative flow cytometry analysis of the expression of NK-cell maturation receptors (CD57, CD127, CD117 and NKG2A) on gated CD56⁺CD3⁻ TINK cells as compared with that on pNK cells from autologous patients and healthy control subjects. Quadrants depicted were set on isotype controls. (B) The frequency of CD57⁺, CD127⁺, CD117⁺ and NKG2A⁺ NK cells within the above-mentioned three NK-cell populations (n = 15; mean±SEM).</p

Kinetics of surface NKp46 expression after influenza vaccination.

<p>(<b>A</b>) FACS analysis of surface NKp46⁺ NK cell percentages in total NK (black), CD56^bright NK (blue) and CD56^dim NK (red) cells from 8 donor PBMCs at the indicated time points. (<b>B</b>) Longitudinal and phenotypic analysis of NK cells. (CD27⁺, NKp44⁺, CXCR6⁺ and CCR7⁺ NK cell frequencies for subjects #1 and #2 were unavailable.)</p

TGF-β1 inhibits the NK cell cycle <i>in vitro</i>.

<p>NK cells from healthy controls were preincubated with IT serum, IA serum, IN serum or anti-TGF-β1Ab. The proportion of cells in the G1 and S phases of the cell cycle was determined using flow cytometry, which demonstrated that TGF-β1 inhibited cell proliferation and induced G1-phase arrest. * P<0.05, ** P<0.01, and *** P<0.001.</p

NKG2D and 2B4 expression is decreased on circulating NK cells during the IT phase of HBV infection.

<p>Immunofluorescent staining of human cells was performed to investigate the expression of NKG2D and 2B4 on NK cells, as gated by FACS analysis. Horizontal bars denote the means. (A) NKG2D and 2B4 expression on total CD3⁺CD56⁻ NK cells within the lymphocyte gate from a representative healthy control or CHB patient. (B and C) Differential NKG2D and 2B4 expression on total NK cells within the lymphocyte gate in HC and samples from patients in the IT, IA and IN phases. (D and E) Absolute counts of NKG2D⁺ and 2B4⁺NK cells in HC, IT, IA and IN. The expression of NKG2D and 2B4 was lower, both by percentage and absolute count, on IT NK cells than on NK cells from both healthy controls and other chronic patients. (F) Inverse correlation between serum 2B4 levels and NKG2D levels on NK cells in HBV patients. Pearson's correlation coefficient: r = 0.7695, P<0.0001. * p<0.05 and *** p<0.001.</p

The kinetics of DN NK-cell accumulation in tumours is associated with tumour progression in vivo.

<p>(A) To establish tumours, C57BL/6 mice were intrapleurally injected with 5×10⁵ live Lewis lung cancer (LLC) cells. Representative flow cytometry analysis of lung TINK cells at different time-points after LLC injection. Dot plots were gated on live NK cells using a lymphocyte gate based on forward scatter versus side scatter and an NK-cell gate using NK1.1⁺CD3⁻ cells. Quadrants depicted were set on isotype controls. (B) Representative flow cytometry analysis of CD27/CD11b expression in lung TINK cells at different time-points after LLC injection. Quadrants depicted were set on isotype controls. (C) The frequency of TINK cells in lung tissue isolated from C57BL/6 mice at each time-point (n = 6 each). (D–F) The frequency of each subset of lung TINK cells isolated from C57BL/6 mice at each time-point (n = 6 each). All experiments were performed three times with similar results.</p

NKp46(S) expression on CD56^dim NK cells after vaccination inversely correlates with IFN-γ secretion to A/PR8 restimulation.

<p>FACS analysis of NKp46 expression (MFI) on CD3⁻CD56^dim NK cells from PBMCs of both vaccinated and control subjects (left y-axis, blue). PBMCs restimulated ex vivo with or without A/PR8 were evaluated at 18 hours for IFN-γ secretion by gating on CD3⁻CD56^dim NK cells (right y-axis, red). NK responsiveness was calculated as: % A/PR8-stimulated IFN-γ⁺CD3⁻CD56^dim NK cells—% control IFN-γ⁺CD3⁻CD56^dim NK cells.</p

Memory NK cell phenotypes and secondary-response features.

<p>(<b>A</b>) NK cells from vaccinated-donor PBMCs were purified at day 0 and then again at 1 month post-vaccination for immunofluorescence assays. Histograms (left) show NKp46 fluorescence intensity on the red lines of cells marked with an asterisk (red). Graphs show NKp46(I) in memory NK cells (1 month post-vaccination) versus NKp46(S) on naive NK cells (day 0). (<b>B</b>) Representative confocal micrographs show differences between naive (day 0) and memory (3 months post-vaccination) cells in terms of NKp46 (green) and IFN-γ (red) expression after A/PR8 restimulation. NKp46 and IFN-γ colocalization is shown in yellow. (<b>C</b>) Representative confocal micrographs of unstimulated control or 18-hour A/PR8-restimulated PBMCs (subjects #9, #10; 1 month post-vaccination) were stained with NKp46 (green) and IFN-γ (red). Red lines indicate cell diameters. (<b>D</b>) Diameter and surface area of NKp46⁺IFN-γ⁺ (red) and NKp46⁺IFN-γ⁻ (blue) cells (n = 45). Mean ± SEM. ***<i>P</i> < 0.005; Student’s <i>t</i>-test. For all micrographs: scale bars, 5 μm; original magnification, 63× oil.</p