CD300a⁺ CD4 T cells are more polyfunctional than CD300a⁻ CD4 T cells.

<p>(A) Purified CD4 T cells were stimulated with PMA and ionomycin for 4–5 h. Then, cells were stained for cell surface expression of CD300a and production of IFN-γ and TNF-α and/or IL-2. The percentage of double and triple producers in the CD300a⁺ and CD300a⁻ cells is shown. Each symbol represents a different donor. (B) Graphic representation of the average ± SEM of the MFI of IFN-γ, IL-2 or TNF-α for the triple producers shown in panel A. (C) Purified CD4 T cells were stimulated with PMA and ionomycin for 4–5 h. Then, cells were stained for cell surface expression of CD300a and the production of the indicated cytokines. The percentage of triple cytokine producers in the CD300a⁺ and CD300a⁻ subsets is shown. Each symbol is a different donor.</p

CD300a⁺ cells up-regulate Eomes after stimulation.

<p>(A) mRNAs for the indicated transcription factors, and IL-12Rβ2 and Jak3 were quantified by real-time PCR. Graph represents the average ± SEM. AU: arbitrary units. Data are from 5–6 donors. (B) Purified CD4 T cells were stimulated with plate bound anti-CD3 plus anti-CD28 mAb for 24 h. The lymphocyte gate was determined according to the forward and side scatter parameters. Then cells were stained for cell surface expression of CD300a and for the intracellular expression of IFN-γ. The expression of Eomes, T-bet and CD300a was determined in the IFN-γ⁺ cells. Results are representative of three donors.</p

Flow cytometric analysis of human peripheral blood CD4 T cells.

<p>In the upper panel, freshly isolated CD4 T cells were labeled with anti-CD4, -CD45RO, and -CD300a mAb. The lymphocyte gate was determined according to the forward and side scatter parameters. Then CD45RO expression was determined for the CD4⁺ cells. The expression of CD300a was assessed in naïve cells (CD45RO⁻, dashed line) and memory cells (CD45RO⁺, continuous line). In the lower panel, freshly isolated CD4 T cells were labeled with anti-CD4, -CD25, -CD127 and -CD300a mAb. After electronically gating the CD4⁺ cells, the expression of CD300a was assessed for the Treg cells (CD25⁺CD127^low, continuous line) and non Treg cells (dashed line). Results are representative of 12 healthy donors.</p

Human circulating IFN-γ producing CD4 T cells are enriched in the CD300a⁺ subset.

<p>(A) Flow cytometric analyses for the production of IFN-γ, IL-4 and IL-17 in combination with cell surface expression of CD300a by purified CD4 T cells stimulated with PMA and ionomycin for 4–5 h. The lymphocyte gate was determined according to the forward and side scatter parameters. The CD300a expression was determined for the cytokine producing cells. A representative healthy donor is shown. The bar graph represents the average ± SEM of the percentage of CD300a⁺ cells within each cytokine producing subset. Results are from 15–19 donors. (B) MFI of IFN-γ expression in the cytokine producing memory CD300a⁺ and CD300a⁻ cells. (C) Expression of CXCR3 and CD300a by circulating memory CD4 T cells. Freshly isolated CD4 T cells were labeled with anti-CD4,-CD45RO, -CD300a and -CXCR3. Dot plots are from CD4⁺CD45RO⁺ gated cells. Results from a representative donor are shown from four donors that were analyzed. (D) PBMCs from healthy donors were stimulated overnight with CMV pp65 or TT in the presence of brefeldin A and then analyzed for the production of IFN-γ and IL-2 by memory CD4 T cells (CD4⁺CD45RO⁺). For the cytokine positive cells, the cell surface expression of CD300a was measured. The response of two of the four donors analyzed is shown.</p

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

TGF-β1 down-regulates the expression of CD300a and Eomes in Th1 cells.

<p>(A) Sorted naïve CD4 T cells were polarized under Th1 or Th17 conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010636#s4" target="_blank">materials and methods</a>. At day 12, cells were stimulated with PMA and ionomycin for 4 h, after which the cell surface expression of CD300a and the production of IFN-γ or IL-17 was determined. Results from a representative donor are shown from four donors that were analyzed. (B) Proliferation, as measured by CFSE dilution, of purified CD4 cells in response to plate bound anti-CD3 plus anti-CD28 mAb in the absence or presence of increasing amounts of TGF-β1. After three days of stimulation cells were rested four additional days. After the resting period, the cell surface expression of CD300a was measured. Results from a representative donor are shown from three donors that were analyzed. (C) Sorted naïve CD4 T cells were polarized under Th1 conditions in the absence or presence of 5 ng/ml of TGF-β1. At day 12, cells were stimulated with PMA and ionomycin for 4 h and the cell surface expression of CD300a and production of IFN-γ were determined. Results from a representative donor are shown from four donors that were analyzed. (D) Sorted naïve CD4 T cells were polarized under Th1 conditions in the absence or presence of 5 ng/ml of TGF-β1. At day 12, cells were stimulated with plate bound anti-CD3 + anti-CD28 mAb overnight, followed by RNA extraction. Specific mRNAs for IFN-γ, T-bet and Eomes were quantified by real-time PCR. Bar graphs show the averages of the percentage decrease in the presence of TGF-β1 ± SEM. Data are from 4 donors.</p

The cytokine and transcription factor signature of CD300a⁺ and CD300a⁻ human memory CD4 T cells.

<p>(A) Purified CD4 T cells were sorted into CD45RO⁺CD300a⁺ (black bars) and CD45RO⁺CD300a⁻ (white bars). RNA was extracted and the levels of mRNAs were determined by real-time PCR. Graphs represent the average ± SEM. AU: arbitrary units. Data are from 5–6 donors. These data correspond to the untreated (none) samples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010636#pone-0010636-g005" target="_blank">Figures 5B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010636#pone-0010636-g006" target="_blank">6A</a>. (B) Purified CD4 T cells were sorted into CD45RO⁺CD300a⁺ and CD45RO⁺CD300a⁻ and then stimulated overnight with plate bound anti-CD3 or anti-CD3 plus anti-CD28 mAb. RNA was extracted from cells and specific cytokine mRNAs were quantified by real-time PCR. (C) Supernatants from the cultures were harvested and the levels of cytokines were measured.</p

Proliferation and phenotype of CD45RO⁺CD300a⁺ and CD45RO⁺CD300a⁻ CD4 T cells.

<p>Proliferation, as measured by CFSE dilution, of sorted CD45RO⁺CD300a⁺ and CD45RO⁺CD300a⁻ cells in response to plate bound anti-CD3 plus anti-CD28 mAb. After three days of stimulation, cells were rested four additional days. After the resting period, the cell surface expression of CXCR3, CD25 and CD300a was measured. IFN-γ production was measured by intracellular staining after a brief stimulation with PMA and ionomycin for 4 h. The bar graph represents the percentage of IFN-γ⁺ cells for each cell generation in the CD300a⁺ and CD300a⁻ subsets. Results shown are representative of four different donors.</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