Differential expression of CD300a/c on human TH1 and TH17 cells

<p>Abstract</p> <p>Background</p> <p>Human memory CD4⁺T cells can be either CD300a/c⁺or CD300a/c^-and subsequent analyses showed that CD4⁺effector memory T (T_EM) cells are mostly CD300a/c⁺, whereas CD4⁺central memory T (T_CM) cells have similar frequencies of CD300a/c⁺and CD300a/c^-cells.</p> <p>Results</p> <p>Extensive phenotypical and functional characterization showed that in both T_CMand T_EMcells, the CD300a/c⁺subset contained a higher number of T_H1 (IFN-γ producing) cells. Alternatively, T_H17 (IL-17a producing) cells tend to be CD300a/c^-, especially in the T_EMsubset. Further characterization of the IL-17a⁺cells showed that cells that produce only this cytokine are mostly CD300a/c^-, while cells that produce IL-17a in combination with other cytokines, especially IFN-γ, are mostly CD300a/c⁺, indicating that the expression of this receptor is associated with cells that produce IFN-γ. Co-ligation of the TCR and CD300a/c in CD4⁺T cells inhibited Ca²⁺mobilization evoked by TCR ligation alone and modulated IFN-γ production on T_H1 polarized cells.</p> <p>Conclusion</p> <p>We conclude that the CD300a/c receptors are differentially expressed on human T_H1 and T_H17 cells and that their ligation is capable of modulating TCR mediated signals.</p

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cγ-1 activity

AbstractWe demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca2+/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cγ1 (PLCγ1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCγ1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCγ1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCγ1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCγ1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed

Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation

Human Th1 Cells That Express CD300a Are Polyfunctional and After Stimulation Up-Regulate the T-Box Transcription Factor Eomesodermin

Human naïve CD4 T cells express low levels of the immunomodulatory receptor CD300a, whereas effector/memory CD4 cells can be either CD300a+ or CD300a−. This suggested that CD300a expression could define a specific subset within the effector/memory CD4 T cell subpopulations. In fact, ex vivo analysis of the IFN-γ producing CD4 T cells showed that they are enriched in the CD300a+ subset. Moreover, stimulated CD4 T cells producing TNF-α and IL-2 besides IFN-γ (polyfunctional) are predominantly CD300a+. In addition to producing markedly higher levels of Th1-associated cytokines, the stimulated CD300a+ CD4 T cells are distinguished by a striking up-regulation of the T-box transcription factor eomesodermin (Eomes), whereas T-bet is up-regulated in both CD300a+ and CD300a− activated CD4 T cells to similar levels. The pleiotropic cytokine TGF-β1 has a determinant role in dictating the development of this Th1 subset, as its presence inhibits the expression of CD300a and down-regulates the expression of Eomes and IFN-γ. We conclude that CD300a+ human Th1 cells tend to be polyfunctional and after stimulation up-regulate Eomes

Assessing climate change associated sea level rise impacts on sea turtle nesting beaches using drones, photogrammetry and a novel GPS system

Climate change associated sea level rise (SLR) is expected to have profound impacts on coastal areas, affecting many species including sea turtles which depend on these habitats for egg incubation. Being able to accurately model beach topography using digital terrain models (DTMs) is therefore crucial to project SLR impacts and develop effective conservation strategies. Traditional survey methods are typically low-cost with low accuracy or high-cost with high accuracy. We present a novel combination of drone-based photogrammetry and a low-cost and portable real-time kinematic (RTK) GPS to create DTMs which are highly accurate (<10 cm error) and visually realistic. This methodology is ideal for surveying coastal sites, can be broadly applied to other species and habitats, and is a relevant tool in supporting the development of Specially Protected Areas. Here we applied this method as a case-study to project three SLR scenarios (0.48, 0.63 and 1.20 m) and assess the future vulnerability and viability of a key nesting habitat for sympatric loggerhead (Caretta caretta) and green turtle (Chelonia mydas) at a key rookery in the Mediterranean. We combined the DTM with 5 years of nest survey data describing location and clutch depth, to identify (1) regions with highest nest densities, (2) nest elevation by species and beach, and (3) estimated proportion of nests inundated under each SLR scenario. On average, green turtles nested at higher elevations than loggerheads (1.8 m vs. 1.32 m, respectively). However, because green turtles dig deeper nests than loggerheads (0.76 m vs. 0.50 m, respectively), these were at similar risk of inundation. For a SLR of 1.2 m, we estimated a loss of 67.3% for loggerhead turtle nests and 59.1% for green turtle nests. Existing natural and artificial barriers may affect the ability of these nesting habitats to remain suitable for nesting through beach migration. This article is protected by copyright. All rights reserved.info:eu-repo/semantics/acceptedVersio

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