Image_1_Implication of Interleukin-12/15/18 and Ruxolitinib in the Phenotype, Proliferation, and Polyfunctionality of Human Cytokine-Preactivated Natural Killer Cells.PDF

<p>A brief in vitro stimulation of natural killer (NK) cells with interleukin (IL)-12, IL-15, and IL-18 endow them a memory-like behavior, characterized by higher effector responses when they are restimulated after a resting period of time. These preactivated NK cells, also known as cytokine-induced memory-like (CIML) NK cells, have several properties that make them a promising tool in cancer immunotherapy. In the present study, we have described the effect that different combinations of IL-12, IL-15, and IL-18 have on the generation of human CIML NK cells. Our data points to a major contribution of IL-15 to CIML NK cell-mediated cytotoxicity against target cells. However, the synergistic effect of the three cytokines grant them the best polyfunctional profile, that is, cells that simultaneously degranulate (CD107a) and produce multiple cytokines and chemokines such as interferon γ, tumor necrosis factor α, and C-C motif chemokine ligand 3. We have also analyzed the involvement of each cytokine and their combinations in the expression of homing receptors CXCR4 and CD62L, as well as the expression of CD25 and IL-2-induced proliferation. Furthermore, we have tested the effects of the Jak1/2 inhibitor ruxolitinib in the generation of CIML NK cells. We found that ruxolitinib-treated CIML NK cells expressed lower levels of CD25 than non-treated CIML NK cells, but exhibited similar proliferation in response to IL-2. In addition, we have also found that ruxolitinib-treated NK cells displayed reduced effector functions after the preactivation, which can be recovered after a 4 days expansion phase in the presence of low doses of IL-2. Altogether, our results describe the impact that each cytokine and the Jak1/2 pathway have in the phenotype, IL-2-induced proliferation, and effector functions of human CIML NK cells.</p

A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells

<div><p>The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.</p></div

Cytokine release by freshly isolated NK cells in the presence of Ab1-10 mAb.

<p>Freshly isolated NK cells were either left untreated or co-cultured with both 293FT cells and M2-293FT cells in the presence and absence of Ab1-10 mAb (Ab). Culture supernatants were harvested and tested for the secretion of human cytokines and chemokines using flow-cytometric bead analysis. The values on the y-axis correspond to the concentrations of TNF-α, IFN-γ, MIP-1α, MIP-1β and RANTES in pg/ml. Graph bars represent the average ± SEM. Data shown are from five independent experiments with freshly isolated NK cells from five donors (* P<0.05, ** P<0.01).</p

The anti-M2e Ab1-10 mAb induces cytokine-preactivated NK cell-mediated ADCC.

<p>293FT or M2-293FT cells were used as targets and control (cultured with low concentrations of IL-15) NK cells (A) and cytokine-preactivated (pretreated with IL-12, IL-15 and IL-18) NK cells (B) were used as effector cells in an ADCC assay. Effector and target cells were co-incubated at different ratios either in the presence or absence of Ab1-10 mAb (Ab). The percentage of specific lysis is shown. Error bars represent the SEM from four independent experiments with NK cells from four donors.</p

Human Ab1-10 mAb recognizes M2e on animal cells.

<p>(A) 293FT cells (left) and M2-293FT cells (right) were cell surface stained with the human anti-M2 Ab1-10 mAb (upper panel) and the mouse anti-M2 14C2 mAb (lower panel) and analyzed by flow cytometry. Shaded histograms represent unstained cells. The binding of the isotype controls (dotted line) and anti-M2e specific mAbs (black line) is shown. (B) Uninfected A549 (left) and influenza infected (right) A549-H1N1 cells were cell surface stained with the human anti-M2 Ab1-10 mAb. The binding of secondary Ab alone (grey histogram) and the binding of Ab1-10 mAb (black line histogram) is shown.</p

The anti-M2e Ab1-10 mAb induces freshly isolated NK cell-mediated ADCC.

<p>(A) Effectors (freshly isolated NK cells) and targets (293FT and M2-293FT cells) were co-incubated at different ratios either in the presence or absence of Ab1-10 mAb (Ab). The percentage of specific lysis is shown. Error bars represent the standard error of the mean (SEM) from five independent experiments with freshly isolated NK cells from five donors (* P<0.05, ** P<0.01). (B) Influenza infected A549 cells were co-cultured with human NK cells from healthy donors in the absence or presence of Ab1-10 mAb (Ab). The percentage of lysis is shown. Error bars represent the SEM from four independent experiments with freshly isolated NK cells from four donors (* P<0.05).</p

Data_Sheet_1_Altered Expression of CD300a Inhibitory Receptor on CD4+ T Cells From Human Immunodeficiency Virus-1-Infected Patients: Association With Disease Progression Markers.PDF

<p>The ability of the CD300a inhibitory receptor to modulate immune cell functions and its involvement in the pathogenesis of many diseases has aroused a great interest in this molecule. Within human CD4+ T lymphocytes from healthy donors, the inhibitory receptor CD300a is differentially expressed among different T helper subsets. However, there are no data about the expression and regulation of CD300a receptor on CD4+ T cells from human immunodeficiency virus (HIV)-1-infected patients. The objective of this study was to investigate the expression of CD300a on CD4+ T cells from HIV-infected patients on suppressive combined antiretroviral therapy (cART) and cART naïve patients. Our results have demonstrated that the expression levels of this inhibitory receptor were higher on CD4+ T cells from HIV-1 infected subjects compared with healthy donors, and that cART did not reverse the altered expression of CD300a receptor in these patients. We have observed an increase of CD300a expression on both PD1+CD4+ and CD38+CD4+ T cells from HIV-1 infected people. Interestingly, a triple positive (CD300a+PD1+CD38+) subset was expanded in naïve HIV-1 infected patients, while it was very rare in healthy donors and patients on cART. Finally, we found a negative correlation of CD300a expression on CD4+ T lymphocytes and some markers associated with HIV-1 disease progression. Thus, our results show that HIV-1 infection has an impact in the regulation of CD300a inhibitory receptor expression levels, and further studies will shed light into the role of this cell surface receptor in the pathogenesis of HIV infection.</p