T-Cell activation: a queuing theory analysis at low agonist density

We analyze a simple linear triggering model of the T-cell receptor (TCR) within the framework of queuing theory, in which TCRs enter the queue upon full activation and exit by downregulation. We fit our model to four experimentally characterized threshold activation criteria and analyze their specificity and sensitivity: the initial calcium spike, cytotoxicity, immunological synapse formation, and cytokine secretion. Specificity characteristics improve as the time window for detection increases, saturating for time periods on the timescale of downregulation; thus, the calcium spike (30 s) has low specificity but a sensitivity to single-peptide MHC ligands, while the cytokine threshold (1 h) can distinguish ligands with a 30% variation in the complex lifetime. However, a robustness analysis shows that these properties are degraded when the queue parameters are subject to variation—for example, under stochasticity in the ligand number in the cell-cell interface and population variation in the cellular threshold. A time integration of the queue over a period of hours is shown to be able to control parameter noise efficiently for realistic parameter values when integrated over sufficiently long time periods (hours), the discrimination characteristics being determined by the TCR signal cascade kinetics (a kinetic proofreading scheme). Therefore, through a combination of thresholds and signal integration, a T cell can be responsive to low ligand density and specific to agonist quality. We suggest that multiple threshold mechanisms are employed to establish the conditions for efficient signal integration, i.e., coordinate the formation of a stable contact interface

Calcium Current Activated by Depletion of Calcium Stores in Xenopus Oocytes

Ca2+ currents activated by depletion of Ca2+ stores in Xenopus oocytes were studied with a two-electrode voltage clamp. Buffering of cytosolic Ca2+ with EGTA and MeBAPTA abolished ICl(Ca) and unmasked a current in oocytes that was activated by InsP3 or ionomycin in minutes and by thapsigargin or the chelators themselves over hours. At −60 mV in 10 mM extracellular CaCl2, the current was typically around −90 or −160 nA in oocytes loaded with EGTA or MeBAPTA, respectively. This current was judged to be a Ca2+-selective current for the following reasons: (a) it was inwardly rectifying and reversed at membrane potentials usually more positive than +40 mV; (b) it was dependent on extracellular [CaCl2] with Km = 11.5 mM; (c) it was highly selective for Ca2+ against monovalent cations Na+ and K+, because replacing Na+ and K+ by N-methyl-d-glucammonium did not reduce the amplitude or voltage dependence of the current significantly; and (d) Ca2+, Sr2+, and Ba2+ currents had similar instantaneous conductances, but Sr2+ and Ba2+ currents appeared to inactivate more strongly than Ca2+. This Ca2+ current was blocked by metal ions with the following potency sequence: Mg2+ << Ni2+ ≈ Co2+ ≈ Mn2+ < Cd2+ << Zn2+ << La3+. It was also inhibited by niflumic acid, which is commonly used to block ICl(Ca). PMA partially inhibited the Ca2+ current, and this effect was mostly abolished by calphostin C, indicating that the Ca2+ current is sensitive to protein kinase C. These results are the first detailed electrophysiological characterization of depletion-activated Ca2+ current in nondialyzed cells. Because exogenous molecules and channels are easy to introduce into oocytes and the distortions in measuring ICl(Ca) can now be bypassed, oocytes are now a superior system in which to analyze the activation mechanisms of capacitative Ca2+ influx

Early T Cell Signalling Is Reversibly Altered in PD-1+ T Lymphocytes Infiltrating Human Tumors

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL

Overexpression of CALNUC (Nucleobindin) Increases Agonist and Thapsigargin Releasable Ca2+ Storage in the Golgi

We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515–1527). In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 μg CALNUC/mg Golgi protein, 2.5 × 105 CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 μM, binding capacity = 1.1 μmol Ca2+/μmol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand α helix from CALNUC completely abolished its Ca2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, α-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store

Agonist-induced calcium entry correlates with STIM1 translocation

The mechanisms of agonist-induced calcium entry (ACE) following depletion of intracellular calcium stores have not been fully established. We report here that calcium-independent phospholipase A (iPLA2) is required for robust Ca2+ entry in HaCaT keratinocytes following ATP or UTP stimulation. Lysophosphatidic acid (LPA), an unrelated agonist, evoked Ca2+ release without inducing robust Ca2+ entry. Both LPA and UTP induced the redistribution of STIM1 into puncta which localized to regions near or at the plasma membrane, as well as within the cytoplasm. Plasma membrane-associated STIM1 remained high for up to 10 min after UTP stimulation, whereas it had returned almost to baseline by that time point in LPA-stimulated cells. This correlated with faster reloading of the endoplasmic reticulum Ca2+ stores in LPA treated cells. Thus by differentially regulating store-refilling after agonist-mediated depletion, LPA and UTP may exert distinct effects on the duration of STIM1 localization at the plasma membrane, and thus, on the magnitude and duration of ACE

EC-18, a Synthetic Monoacetyldiacylglyceride, Inhibits Hematogenous Metastasis of KIGB-5 Biliary Cancer Cell in Hamster Model

EC-18 (monoacetyldiacylglyceride) stimulates T cell production of IL-2, IL-4, IL-12, IFN-γ, and GM-CSF in vitro. To study the effects of these cytokines stimulated by EC-18 on cancer cells, we applied hamster biliary cancer model, a difficult cancer to treat. Cancer (KIGB-5) cells were given intravenously to produce hematogenous metastatic lung lesions which were treated with EC-18 at 10, 25, and 50 mg/kg/day respectively. The fourth group was untreated control. At 4th, 8th, and 12th week the lungs were examined. EC-18 treated groups showed only a few microscopic lung lesions and no evidence of metastatic lesion with highest dose whereas widespread gross lung lesions were observed in untreated control. To investigate whether the anti-tumor effect of EC-18 is associated with suppression of tumor cell Toll-like receptor 4 (TLR-4) expression in addition to stimulation of the immune cells, KIGB-5 cells were exposed to LPS with or without EC-18. TLR-4 mRNA and protein expression, measured by reverse transcriptase PCR (RT-PCR), real-time quantitative PCR and western blot analysis, showed suppression of TLR-4 expression in KIGB-5 cells treated with EC-18 compared with control. In conclusion, EC-18 has a significant anti-tumor effect in this experimental model of biliary cancer suggesting potential for clinical application to this difficult cancer

Prolonged calcium influx after termination of light-induced calcium release in invertebrate photoreceptors

© The Authors, 2009 . This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of General Physiology 134 (2009): 177-189, doi:10.1085/jgp.200910214.In microvillar photoreceptors, light stimulates the phospholipase C cascade and triggers an elevation of cytosolic Ca2+ that is essential for the regulation of both visual excitation and sensory adaptation. In some organisms, influx through light-activated ion channels contributes to the Ca2+ increase. In contrast, in other species, such as Lima, Ca2+ is initially only released from an intracellular pool, as the light-sensitive conductance is negligibly permeable to calcium ions. As a consequence, coping with sustained stimulation poses a challenge, requiring an alternative pathway for further calcium mobilization. We observed that after bright or prolonged illumination, the receptor potential of Lima photoreceptors is followed by the gradual development of an after-depolarization that decays in 1–4 minutes. Under voltage clamp, a graded, slow inward current (Islow) can be reproducibly elicited by flashes that saturate the photocurrent, and can reach a peak amplitude in excess of 200 pA. Islow obtains after replacing extracellular Na+ with Li+, guanidinium, or N-methyl-D-glucamine, indicating that it does not reflect the activation of an electrogenic Na/Ca exchange mechanism. An increase in membrane conductance accompanies the slow current. Islow is impervious to anion replacements and can be measured with extracellular Ca2+ as the sole permeant species; Ba can substitute for Ca2+ but Mg2+ cannot. A persistent Ca2+ elevation parallels Islow, when no further internal release takes place. Thus, this slow current could contribute to sustained Ca2+ mobilization and the concomitant regulation of the phototransduction machinery. Although reminiscent of the classical store depletion–operated calcium influx described in other cells, Islow appears to diverge in some significant aspects, such as its large size and insensitivity to SKF96365 and lanthanum; therefore, it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors.This work was supported by National Science Foundation grant 0639774

A Novel ZAP-70 Dependent FRET Based Biosensor Reveals Kinase Activity at both the Immunological Synapse and the Antisynapse

Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCγ1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or “antisynapse”