Prolonged contact with dendritic cells turns lymph node‐resident NK cells into anti‐tumor effectors

Abstract Natural killer (NK) cells are critical players against tumors. The outcome of anti‐tumor vaccination protocols depends on the efficiency of NK‐cell activation, and efforts are constantly made to manipulate them for immunotherapeutic approaches. Thus, a better understanding of NK‐cell activation dynamics is needed. NK‐cell interactions with accessory cells and trafficking between secondary lymphoid organs and tumoral tissues remain poorly characterized. Here, we show that upon triggering innate immunity with lipopolysaccharide (LPS), NK cells are transiently activated, leave the lymph node, and infiltrate the tumor, delaying its growth. Interestingly, NK cells are not actively recruited at the draining lymph node early after LPS administration, but continue their regular homeostatic turnover. Therefore, NK cells resident in the lymph node at the time of LPS administration become activated and exert anti‐tumor functions. NK‐cell activation correlates with the establishment of prolonged interactions with dendritic cells (DCs) in lymph nodes, as observed by two‐photon microscopy. Close DC and NK‐cell contacts are essential for the localized delivery of DC‐derived IL‐18 to NK cells, a strict requirement in NK‐cell activation

Accumulative Difference Image Protocol for Particle Tracking in Fluorescence Microscopy Tested in Mouse Lymphonodes

The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done

IL-15 cis Presentation Is Required for Optimal NK Cell Activation in Lipopolysaccharide-Mediated Inflammatory Conditions

Natural killer (NK) cells have antitumor, antiviral, and antibacterial functions, and efforts are being made to manipulate them in immunotherapeutic approaches. However, their activation mechanisms remain poorly defined, particularly during bacterial infections. Here, we show that upon lipopolysaccharide or E. coli exposure, dendritic cells (DCs) produce three cytokines—interleukin 2 (IL-2), IL-18, and interferon β (IFN-β)—necessary and sufficient for NK cell activation. IFN-β enhances NK cell activation by inducing IL-15 and IL-15 receptor α not only in DCs but, surprisingly, also in NK cells. This process allows the transfer of IL-15 on NK cell surface and its cis presentation. cis-presented NK cell-derived and trans-presented DC-derived IL-15 contribute equally to optimal NK cell activation

Circulating miR-150 modulation in human serum upon flu vaccination.

<p>A. miR-150 quantities relative to exogenous spike-in ath miR-159a in sera of 50 H1N1-MF59 vaccinated children (samples collected at time of first dose, T0, at time of second dose 30 days after, T1 and 30 days after the second dose, T2) (left) and 46 pairs of samples (time of vaccination, T0 and 30 days after, T1) from H1N1-MF59 vaccinated healthy adults (right). Data were centered on the mean at T0 and mean values, SEM and two-tailed paired t test p values are reported. B. Box plot of miR-150 quantities relative to exogenous spike-in ath miR-159a (whiskers: 10-90 percentile) in the indicated serum compartments of 17 pairs of H1N1-MF59 at T0 (white) and T1 (grey). Two-tailed paired t test p values are reported. C. Receiver Operating Characteristic (ROC) curves for total serum, nanovesicular and microvesicular miR-150 increment in H1N1-MF59 vaccinated adults compared to pre-vaccination level (SE=Sensitivity; SP= Specificity). Area under the curve (AUC) and p value (calculated with χ² test) for nanovesicular miR-150 are reported.</p

Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation

<div><p>Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation <i>in vitro</i>, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. <i>In vivo</i>, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.</p> </div

miR-150 expression in human resting lymphocytes and tissues.

<p>A. Box plot of miRNome relative quantities in 17 different lymphocyte subsets, as indicated (blue, B; red, CD4⁺ T; green, CD8⁺ T; grey, NK). Only co-expressed miRNAs with a Ct<35 were considered. White circles indicate miR-150 expression level. B. miR-150 level in a panel of 20 different human tissues (as indicated) by RT-qPCR, relative to the internal control MammU6, and reported in percentage relative expression among tissues.</p