Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells.

The reorientation of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell enables the directional secretion of cytokines and lytic factors. By single-cell photoactivation of the T cell antigen receptor, we show that MTOC polarization is driven by localized accumulation of diacylglycerol (DAG). MTOC reorientation was closely preceded first by production of DAG and then by recruitment of the microtubule motor protein dynein. Blocking DAG production or disrupting the localization of DAG impaired MTOC recruitment. Localized DAG accumulation was also required for cytotoxic T cell-mediated killing. Furthermore, photoactivation of DAG itself was sufficient to induce transient polarization. Our data identify a DAG-dependent pathway that signals through dynein to control microtubule polarity in T cells.

NK cell tolerance of self-specific activating receptor KIR2DS1 in individuals with cognate HLA-C2 ligand.

NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC class I Ags and protect healthy cells from NK cell-mediated autoaggression. However, certain activating receptors, including the human activating killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC class I. This fact raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti-HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of the presence or absence of HLA-C2 ligand in the donor. The frequency of anti-HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, no significant difference was seen in the frequency of anti-HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2, compared with all other donors, had significantly reduced frequency of anti-HLA-C2 reactive clones. The 2DS1 positive clones that express inhibitory KIR for self-HLA class I were commonly noncytotoxic, and anti-HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti-HLA-C2 reactivity were also present posttransplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti-HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2.

Protein kinase C-θ clustering at immunological synapses amplifies effector responses in NK cells.

In lymphocytes, stimulation of cell surface activating receptors induces the formation of protein microclusters at the plasma membrane that contain the receptor itself, along with other signaling molecules. Although these microclusters are generally thought to be crucial for promoting downstream cellular responses, evidence that specifically links clustering potential to signaling output is lacking. We found that protein kinase C-θ (PKCθ), a key signaling molecule in multiple lymphocyte subsets, formed microclusters in activated NK cells. These microclusters coalesced within the immunological synapse between the NK cell and its target cell. Clustering was mediated by the regulatory region of PKCθ and specifically required a putative phosphotyrosine-binding site within its N-terminal C2 domain. Whereas expression of wild-type PKCθ rescued the cytokine production defect displayed by PKCθ-deficient NK cells, expression of a PKCθ point-mutant incapable of forming microclusters had little to no effect. Hence, PKCθ clustering was necessary for optimal effector function. Notably, only receptors containing ITAMs induced PKCθ microclusters on their own, explaining previous observations that ITAM-coupled receptors promote stronger activating signals and effector responses than do receptors lacking these motifs. Taken together, our results provide a cell biological basis for the role of PKCθ clustering during NK cell activation, and highlight the importance of subcellular compartmentalization for lymphocyte signal transduction.

Diacylglycerol kinases: at the hub of cell signalling.

DGKs (diacylglycerol kinases) are members of a unique and conserved family of intracellular lipid kinases that phosphorylate DAG (diacylglycerol), catalysing its conversion into PA (phosphatidic acid). This reaction leads to attenuation of DAG levels in the cell membrane, regulating a host of intracellular signalling proteins that have evolved the ability to bind this lipid. The product of the DGK reaction, PA, is also linked to the regulation of diverse functions, including cell growth, membrane trafficking, differentiation and migration. In multicellular eukaryotes, DGKs provide a link between lipid metabolism and signalling. Genetic experiments in Caenorhabditis elegans, Drosophila melanogaster and mice have started to unveil the role of members of this protein family as modulators of receptor-dependent responses in processes such as synaptic transmission and photoreceptor transduction, as well as acquired and innate immune responses. Recent discoveries provide new insights into the complex mechanisms controlling DGK activation and their participation in receptor-regulated processes. After more than 50 years of intense research, the DGK pathway emerges as a key player in the regulation of cell responses, offering new possibilities of therapeutic intervention in human pathologies, including cancer, heart disease, diabetes, brain afflictions and immune dysfunctions.

Eficacia de la radioterapia en los pacientes con cáncer laríngeo en estadio I y II / Efficacy of radiotherapy in patients with laryngeal cancer in stage I and II

RESUMEN Fundamento: el cáncer de laringe es una neoplasia maligna de cabeza y cuello frecuente, el tipo histológico predominante es el carcinoma epidermoide y la mayoría de los afectados, presentan por lo general el tumor localizado en la región glótica, predomina en hombres en una relación de 4:1, el incremento aunque existen otros factores está asociado al mayor consumo de tabaco y alcohol. Objetivo: evaluar la eficacia de la radioterapia a los cinco años de finalizado el tratamiento, como método terapéutico en pacientes con cáncer laríngeo en estadio I y II. Métodos: se realizó un estudio descriptivo, longitudinal, retrospectivo en 27 pacientes, con el propósito de evaluar la eficacia de la radioterapia como método terapéutico en pacientes diagnosticados con cáncer laríngeo en etapa I y II en la provincia Camagüey, desde enero de 2014 hasta enero de 2017. Resultados: la edad y el sexo no influyeron en la eficacia del tratamiento. Los pacientes en un estadio I evolucionaron mejor a la radioterapia. La región anatómica más frecuente como sitio de implantación del tumor fue la glotis, sin embargo, dicho parámetro no jugó un papel fundamental ya que la radioterapia fue eficaz en el resto de los grupos histopatológicos. Los pacientes con carcinoma epidermoide indiferenciado, respondieron todos de forma satisfactoria a la radioterapia, a pesar que en sentido general e independientemente el grupo histopatológicos los resultados fueron satisfactorios. La mucositis y la anemia fueron las complicaciones más frecuentes, las mismas no afectaron la eficacia de la radiación. Conclusiones: la terapia local garantiza una excelente calidad de vida en los enfermos, por lo que se recomienda capacitar al personal de salud de las diferentes áreas de atención, para garantizar un diagnóstico precoz del cáncer laríngeo, lo que permitirá una terapéutica en estadios tempranos y mayor supervivencia.

ABSTRACT Background: Laryngeal cancer is a frequent malignant neoplasm of the head and neck, the predominant histological type is squamous cell carcinoma and the majority of those affected by this histological type usually present the tumor located in the glottal region, predominantly in men in a 4: 1 ratio, the increase although there are other factors is associated with greater consumption of tobacco and alcohol. Objective: to evaluate the efficacy of radiotherapy five years after the end of treatment, as a therapeutic method in patients with stage I and II laryngeal cancer. Methods: descriptive, longitudinal, retrospective study was carried out in 27 patients, with the purpose of evaluating the efficacy of radiotherapy as a therapeutic method in patients diagnosed with stage I and II laryngeal cancer in the province of Camagüey, in the period from January 2014 to January 2017. Results: age and sex did not influence the efficacy of the treatment. Patients in a stage I evolved better to radiotherapy. The most frequent anatomical region as site of implantation of the tumor was the glottis, however, this parameter did not play a fundamental role since radiotherapy was effective in the rest of the histopathological groups. The patients with undifferentiated squamous cell carcinoma all responded satisfactorily to radiotherapy, although in general and independently the histopathological group the results were satisfactory. Mucositis and anemia were the most frequent complications, they did not affect the effectiveness of the radiation. Conclusions: considering that local therapy guarantees an excellent quality of life in patients, it is recommended to train the health personnel of the different areas of care, to guarantee an early diagnosis of laryngeal cancer, which will allow a therapy in early stages and greater survival.

Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.

Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell-activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.

Lck-dependent tyrosine phosphorylation of diacylglycerol kinase alpha regulates its membrane association in T cells.

TCR engagement triggers phospholipase Cgamma1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca(2+), both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase alpha (DGKalpha) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKalpha to the membrane have not been fully elucidated, although Ca(2+) influx and tyrosine kinase activation were proposed to be required. We show that DGKalpha is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKalpha during TCR activation and shows that pY335DGKalpha is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKalpha function and suggest a mechanism by which Lck-dependent phosphorylation and Ca(2+) elevation regulate DGKalpha membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKalpha relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.

Role of the diacylglycerol kinase alpha-conserved domains in membrane targeting in intact T cells.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement.

Directional cell movement in response to external chemical gradients requires establishment of front-rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform beta (PIPKIbeta) has a role in organizing signaling at the cell rear. We found that PIPKIbeta polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIbeta localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIbeta C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIbeta with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIbeta in these processes.

Diacylglycerol kinase alpha regulates the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes.

Fas ligand (FasL) mediates both apoptotic and inflammatory responses in the immune system. FasL function critically depends on the different forms of FasL; soluble Fas ligand lacking the transmembrane and cytoplasmic domains is a poor mediator of apoptosis, whereas full-length, membrane-associated FasL (mFasL) is pro-apoptotic. mFasL can be released from T lymphocytes, via the secretion of mFasL-bearing exosomes. mFasL in exosomes retains its activity in triggering Fas-dependent apoptosis, providing an alternative mechanism of cell death that does not necessarily imply cell-to-cell contact. Diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, is involved in the attenuation of DAG-derived responses initiated at the plasma membrane that lead to T lymphocyte activation. Here we studied the role of DGKalpha on activation-induced cell death on a T cell line and primary T lymphoblasts. The inhibition of DGKalpha increases the secretion of lethal exosomes bearing mFas ligand and subsequent apoptosis. On the contrary, the overactivation of the DGKalpha pathway inhibits exosome secretion and subsequent apoptosis. DGKalpha was found associated with the trans-Golgi network and late endosomal compartments. Our results support the hypothesis that the DGKalpha effect on apoptosis occurs via the regulation of the release of lethal exosomes by the exocytic pathway, and point out that the spatial orchestration of the different pools of DAG (plasma membrane and Golgi membranes) by DGKalpha is crucial for the control of cell activation and also for the regulation of the secretion of lethal exosomes, which in turn controls cell death.

Importance of chroman ring and tyrosine phosphorylation in the subtype-specific translocation and activation of diacylglycerol kinase alpha by D-alpha-tocopherol.

Diacylglycerol kinase (DGK) has been suggested to be a pharmacological target of D-alpha-tocopherol for diabetic renal dysfunctions. However, the DGK subtypes involved in the D-alpha-tocopherol-induced improvement of diabetic renal dysfunctions and the activation mechanisms of DGK by D-alpha-tocopherol are still unknown. Therefore, using GFP-tagged DGKalpha, beta, gamma, epsilon and zeta, we analyzed their response to D-alpha-tocopherol and its derivatives. Only DGKalpha was translocated from the cytoplasm to the plasma membrane with elevation of kinase activity. In addition, troglitazone and trolox possessing 'chroman ring' similarly to D-alpha-tocopherol, induced the subtype-specific translocation of DGKalpha. Furthermore, the translocation of DGKalpha was abolished by pretreatment with tyrosine kinase inhibitors or by mutation on Tyr-334 of the kinase (YF mutant). D-alpha-tocopheryl succinate enhanced the Src-mediated tyrosine phosphorylation of wild-type DGKalpha but the same reagent did not enhance that of the YF mutant. These results demonstrate that tyrosine phosphorylation on Tyr-334 and chroman ring are important for the D-alpha-tocopherol-induced translocation of DGKalpha.

Regulation of diacylglycerol kinase alpha by phosphoinositide 3-kinase lipid products.

Diacylglycerol kinase alpha (DAGK alpha), like all type I DAGKs, has calcium regulatory motifs that act as negative regulators of enzyme activity and localization. Accordingly, DAGK alpha is activated by phospholipase C-coupled receptors in a calcium-dependent manner. One of the first functions attributed to DAGK alpha in lymphocytes was that of regulating interleukin 2-induced cell cycle entry. Interleukin-2 nonetheless exerts its action in the absence of cytosolic calcium increase. We have studied alternative receptor-derived signals to explain calcium-independent DAGK alpha activation, and show that DAGK alpha is stimulated by Src-like kinase-dependent phosphoinositide 3 kinase (PI3K) activation in lymphocytes. Our results demonstrate that, in vivo, the increase in cellular levels of PI3K products is sufficient to induce DAGK alpha activation, allowing DAGK alpha relocation to the intact lymphocyte plasma membrane. This activation is isoform-specific, because other type I DAGKs are not subject to this type of regulation. These studies are the first to describe a pathway in which, in the absence of receptor-regulated calcium increase, DAGK alpha activation and membrane localization is a direct consequence of PI3K activation.