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

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”

The Scaffolding Protein Dlg1 Is a Negative Regulator of Cell-Free Virus Infectivity but Not of Cell-to-Cell HIV-1 Transmission in T Cells

Background: Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Methodology/Principal Findings: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA

Modulation de la permeabilite jonctionnelle par l'acetylcholine dans les glandes lacrymales de rat

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Light-tunable optical cell manipulation via photoactive azobenzene-containing thin film bio-substrate

In-vivo, real-time study of the local and collective cellular biomechanical responses requires the fine and selective manipulation of the cellular environment. One innovative pathway is the use of photoactive bio-substrates such as azobenzene-containing materials (azopolymers), with optically tunable properties. In this work we show an innovative simple method to optically stimulate cells locally, by light-excitation of an azo-polymer derivative bio-substrate. Excited cells exhibit spectacular motility and reversible area shrinkage, which is dependent on the illumination. The photomechanical mechanisms taking place at the substrate and the cell/environment mechanical phenomena require further investigation

Light‐tunable optical cell manipulation via photoactive azobenzene‐containing thin film bio‐substrate

International audienceIn-vivo, real-time study of the local and collective cellular biomechanical responses requires the fine and selective manipulation of the cellular environment. One innovative pathway is the use of photoactive bio-substrates such as azobenzene-containing materials, which exhibit spectacular photomechanical properties, to optically trigger the local, mechanical stimulation of cells. Excited cells exhibit spectacular morphological modifications and area shrinkage, which are dependent on the illumination. This demonstrates the capabilities of photomechanically active substrates to study the phenomena resulting from the mechanical interaction of cells with their environment

In-vivo, in-situ, light-tunable manipulation of cells' biomechanics on a photoactive azobenzene bio-substrate

International audienceIn-vivo, real-time study of the local and collective cellular biomechanical responses requires the fine and selective control of the cellular environment. Optical manipulation provides a suitable pathway to achieve non-contact, selective, local, temporal and spatial stimuli. The spectacular photomechanical properties of photoactive bio-substrates such as azobenzene-containing thin polymer films are a new promising strategy to achieve optically triggered local mechanical stimulation of cells. Excited cells exhibit spectacular morphological modifications and area shrinkage, which are dependent on the illumination. In this work we demonstrate that the capabilities of photomechanically active azocontaining substrates to optically stimulate cells' mechanical response can be strongly influenced by the adhesion binding agent used to deposit the living cells on the photoactive layer. This provides a further tool for the photomechanical control of the cellular environment and of the cellular response

Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

International audienceT cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4 + T cell functions are affected by stiffness within the physiological Young's modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/ CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly