Control Of Cell Activity Research Articles

Effectiveness of Soluble CTLA-4-Fc in the Inhibition of Bone Marrow T-Cell Activation in Context of Indoleamine 2.3-Dioxygenase (IDO) and CD4+Foxp3+ Treg Induction.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4+Foxp3+ Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery. Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4+CD69+ and CD4+Foxp3+ percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls. We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4+ T cells activation without influencing CD8+ cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls. This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA.

Abstract 3161: Preparation of aclinical trial with a-TIGIT antagonist antibody EOS884448, which demonstrates potent preclinical activity and safe toxicology profile

Abstract T cell Immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory receptor expressed by lymphocytes, preferentially CD8+, NK, as well as by regulatory T cells (Treg). CD226 (DNAM-1), a co-stimulatory receptor also expressed on NK and T cells, competes with TIGIT for their common ligand, CD155, binding but with a lower affinity. Co-expression of TIGIT and CD226 receptors on T and NK effector cells suggests a role of these molecules in the fine control of cell activation. In cancer, TIGIT expression is upregulated on conventional and even more on regulatory T cells and co-expression with exhaustion markers such as PD-1 is frequent, giving a strong rationale for blocking TIGIT as a therapeutic approach to reverse T or NK cell dysfunction linked with cancer progression. We have developed EOS884448, a fully human antagonist anti-TIGIT antibody that demonstrates strong potential in vitro to compete with TIGIT natural ligands and to prevent CD155-mediated inhibition of T cells from healthy volunteers and cancer patients. In addition, EOS884448 preferentially depletes Treg via an ADCC-triggered mechanism. In vivo, in mouse tumor models, a surrogate a-TIGIT antibody has shown potent antitumor activity through a combination of its antagonistic properties and its effector function through Fc gamma receptor engagement. EOS884448 was tested in a non-human primate GLP toxicology study and demonstrated a safe profile at all tested doses (≤10mg/kg) after repeated injection for up to 4 weeks at weekly intervals (5 doses in total). Pharmacokinetic data showed dose-dependent increase of EOS884448 concentration in the blood that correlated with gradual increased in TIGIT receptor occupancy measured on NK and T cells and that reached saturation in Cynomolgus monkeys dosed at 1mg/kg. In anticipation of the first-in-human (FIH) study of EOS884448, assays to measure PK, ADA and receptor occupancy in human blood samples were developed. In addition, to support the clinical strategy, multiple cancer indications were tested for expression of members of the TIGIT pathway. The frequency of TIGIT-expressing immune cells, analysed by FACS, was increased in PBMCs from cancer patients as compared to healthy donors. Further increase in TIGIT expression (both in proportion and density) was measured on TILs, and particularly on Treg cells that had the highest expression. CD155 expression analysed by IHC in solid cancers (n=284) was higher in tumor and seen in the nine tested indications, with close to 50% of CD155high tumor cells measured in indications including pancreatic, prostate, kidney, gastric and colon cancers. CD226 expression was confirmed by IHC on immune infiltrate in solid tumor samples (n=307). Altogether, preclinical, toxicology and translational medicine data support the initiation of a FIH study of a-TIGIT mAb EOS884448 in cancer patients. Citation Format: Thi Lien-Anh Nguyen, Julia Cuende, Julie Preillon, Lucile Garnero, Virginie Rabolli, Noémie Wald, Catherine Hoofd, Patricia Chevron, Marjorie Mercier, Olivier De Henau, Véronique Bodo, Gregory Driessens. Preparation of aclinical trial with a-TIGIT antagonist antibody EOS884448, which demonstrates potent preclinical activity and safe toxicology profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3161.

Transfer of optogenetic vectors into the brain of neonatal animals to study neuron functions during subsequent periods of development

Optogenetics, that is, the control of cell activity using photosensitive ion channels, opsins, with the light of a specific wavelength, is increasingly being used to study the activities and functions of neurons. The expression of opsins in the cell membrane, making the cell sensitive to light, is achieved by means of the viral vectors, mainly constructed using lentiviruses or adeno-associated viruses (AAVs), with the inserted nucleotide sequences encoding the photochannel proteins. The specific promoter for the target cell type added to the transgene expression cassette provides opsin production only in these target cells. The aim of this work was to briefly describe the optogenetic method, as well as to analyze the possibility to use administration of viral vectors into the brain of neonatal animals to study the function of neurons in vivo during subsequent periods of development. In this analysis, 3-day-old rat pups are intracerebroventricularly injected with the optovector (pAAV-CAMKIIa-ChR2h134-YFP) coding for a neuron-activating photochannel and a marker, yellow fluorescent protein, under the CAMKIIa promoter, specific for glutamatergic neurons. The peak expression of the transferred gene is usually achieved 3–5 weeks after the vector transfer, which is also observed in our experiments. Stimulation of the hippocampal neurons with blue light in the 20-day-old animals injected with the optovector on 3rd day of their lives increases the discharge activity of these neurons, as well as the expression of c-Fos protein, a well-known marker of neuronal activation. The same experiments with older animals (60 days after the neonatal optochannel gene transfer) demonstrate neither any noticeable expression nor photoactivation of the target hippocampal neurons. Thus, the neonatal administration of a viral vector carrying an optochannel gene is suitable for the study of brain neurons in juvenile rats but requires additional assessment of the corresponding gene expression during subsequent periods of development.

Transfer of optogenetic vectors into the brain of neonatal animals to study neuron functions during subsequent periods of development

Optogenetics, that is, the control of cell activity using light-sensitive ion channels opsins with light of a specific wavelength, is increasingly being used to study activities and functions of neurons. Expression of opsins in the cell membrane, followed by the acquisition by the cell of the sensitivity to light is achieved by means of viral vectors, often created on the basis of lentiviral or adeno-associated (AAV) viruses bearing the nucleotide sequence encoding the photo-channel proteins. Inclusion of the cell-specific promoter of interest into the transgene-expression cassette allows opsin to be produced only in the target cells. The aim of this work was to briefly describe the optogenetic method, as well as to analyze the possibility to use administration of viral vectors into the brain of neonatal animals to study the function of neurons in vivo during subsequent periods of development. In this analysis, 3-day-old rat pups received intracerebroventricular injections of optovector (pAAV-CAMKIIa-ChR2h134-YFP), coding for a photo channel, which activates neurons, and the yellow fluorescent marker protein under the CAMKIIa promoter specific for glutamatergic neurons under cold anesthesia. The peak expression of the transferred gene is usually achieved at week 3–5 after the transfer of the vector, which is what was also observed in our experiments. Stimulation of the hippocampal neurons with blue light in the 20-day-old animals, to which opto-vector was transferred at the 3rd day of life, increased the discharge activity of these neurons. This light stimulation increased expression of the recognized marker of neuronal activation protein c-Fos in these photosensitive cells too. The same experiments with older animals, 60 days after the neonatal opto-channel gene transfer, revealed no noticeable expression of this channel or photoactivation of target neurons of the hippocampus. Thus, neonatal administration of a viral vector carrying an opto-channel gene is suitable for the study of brain neurons in rats of juvenile age, and requires additional control for gene expression during subsequent periods of development.

Abstract 38: Simultaneous Optical Pacing and Optical Voltage Mapping in Optogenetic Neonatal Rat Ventricular Myocyte Cultures

Optogenetics is an emerging technology allowing remote and precise control of cell activity in living tissues. Despite its rapid advancements, application of this innovative technology to cardiovascular research is still limited, in part due to shortage of optogenetic cardiac tissue models and compatible imaging methods. The present study aimed to develop an optogenetic culture model using neonatal rat ventricular myocytes (NRVM) expressing light-gated Channelrhodopsin-2 (ChR2) and characterize activation spread during optical stimulation using optical mapping of membrane potential (Vm). Primary NVRM cultures were infected with lentivirus containing ChR2 gene. Cultures were paced electrically or optically with pulses of blue (470 nm) LED light. Activation spread was simultaneously mapped using Vm-sensitive dye (RH-237) and a photodiode mapping system. Results showed that ChR2 could be readily transduced to NRVMs by the lentiviral method; however, high-level ChR2 expression was associated with substantial cell toxicity. Lower ChR2 expression, achieved by administration of bromodeoxyuridine, had minor effects on cell morphology and function while allowing optical pacing at frequencies of 0.5-3 Hz. Simultaneous Vm mapping showed that conduction velocity, APD80, and dV/dtmax were similar in optogenetic and control cultures. Finally, the optogenetic cultures could be optically paced at multiple sites, leading to significantly reduced overall activation time. In summary, we demonstrated that ChR2 expression can cause cell toxicity in NRVM cultures but the toxicity can be mitigated allowing optical pacing and simultaneous optical activation mapping without significant impairment of electrophysiological function. This optogenetic cardiac culture model expands the availability of optogenetic tools for cardiac research.

Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro

BackgroundThe interfacial molecular mechanisms that regulate mammalian cell growth and differentiation have important implications for biotechnology (production of cells and cell products) and medicine (tissue engineering, prosthetic implants, cancer and developmental biology). We demonstrate here that engineered protein motifs can be robustly displayed to mammalian cells in vitro in a highly controlled manner using a soluble protein scaffold designed to self assemble on a gold surface.ResultsA protein was engineered to contain a C-terminal cysteine that would allow chemisorption to gold, followed by 12 amino acids that form a water soluble coil that could switch to a hydrophobic helix in the presence of alkane thiols. Bioactive motifs from either bone morphogenetic protein-2 or osteopontin were added to this scaffold protein and when assembled on a gold surface assessed for their ability to influence cell function. Data demonstrate that osteoblast adhesion and short-term responsiveness to bone morphogenetic protein-2 is dependent on the surface density of a cell adhesive motif derived from osteopontin. Furthermore an immobilised cell interaction motif from bone morphogenetic protein supported bone formation in vitro over 28 days (in the complete absence of other osteogenic supplements). In addition, two-dimensional patterning of this ligand using a soft lithography approach resulted in the spatial control of osteogenesis.ConclusionThese data describe an approach that allows the influence of immobilised protein ligands on cell behaviour to be dissected at the molecular level. This approach presents a durable surface that allows both short (hours or days) and long term (weeks) effects on cell activity to be assessed. This widely applicable approach can provide mechanistic insight into the contribution of immobilised ligands in the control of cell activity.

Incorporation of Photosynthetic Reaction Centers in the Membrane of Human Cells: Toward a New Tool for Optical Control of Cell Activity

The Photosystem I (PSI) reaction center is a photosynthetic membrane complex in which light-induced charge separation is accompanied by the generation of an electric potential. It has been recently proposed as a means to confer light sensitivity to cells possessing voltage-activated ion channels, but the feasibility of heterologous incorporation has not been demonstrated. In this work, methods of delivery and detection of PSI in the membrane of human cells are presented. Purified fractions of PSI were reconstituted in proteoliposomes that were used as vehicles for the membrane incorporation. A fluorescent impermeable dye was entrapped in the vesicles to qualitatively analyze the nature of the vesicle–cell interaction. After incorporation, the localization and orientation of the complexes in the membrane was studied using immuno-fluorescence microscopy. The results showed complexes oriented as in native membranes, which were randomly distributed in clusters over the entire surface of the cell. Additionally, analysis of cell viability showed that the incorporation process does not damage the cell membrane. Taken together, the results of this work suggest that the mammalian cellular membrane is a reasonable environment for the incorporation of PSI complexes, which opens the possibility of using these molecular photovoltaic structures for optical control of cell activity.

Osteogenic differentiation of ES cell-derived EBs mediated by embedded BMP-2 and TGF-beta-1 in a polyelectrolyte multilayer film

ABSTRACTIn recent years, considerable effort has been devoted to the design and controlled fabrication of structured materials with functional properties. The layer by layer buildup of polyelectrolyte multilayer films (PEM films) from oppositely charged polyelectrolytes1 offers new opportunities for the preparation of functionalized biomaterial coatings. This technique allows the preparation of supramolecular nano-architectures exhibiting specific properties in terms of control of cell activation and may also play a role in the development of local drug delivery systems. Peptides, proteins or DNA, chemically bound to polyelectrolytes, adsorbed or embedded in PEM films, have been shown to retain their biological activities. Recently, tissue engineering has merged with stem cell technology with interest to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries disorders because of the low self-regenerating capacity of the matrix secreting cells. We present here for the first time that embedded BMP-2 and TGFβ1 in a multilayered polyelectrolyte film can drive embryonic stem cells to the cartilage or bone differentiation depending on supplementary co-factors. We selected a model system made from layer by layer poly-ℓ-glutamic acid (PℓGA) and poly-ℓ-lysine succinylated (PℓLs) films into which BMP-2 and TGFβ1 have been embedded. Our results demonstrate clearly that we are able to induce osteogenesis in embryonic stem cells mediated by growth factors embedded in a polyelectrolyte multilayer film.

Long-term culture in dexamethasone unmasks an abnormal phenotype in osteoblasts isolated from osteoporotic subjects

We have shown that osteoblastic cells derived from trabecular bone explants of osteoporotic subjects (OP cells) exhibited an altered alkaline phosphatase (ALP) response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] compared to control (CON) cells. Our hypothesis that OP cells have other intrinsic abnormalities was investigated using our cell models representing two different stages of differentiation. OP and CON cells were cultured in the absence (-DEX) or presence (+DEX) of 10 nM dexamethasone (DEX) in 10% fetal calf serum (FCS) prior to exposure to serum-free medium containing 1 nM of PTH and/or 17-beta estradiol (E2). Both OP and CON cells responded to DEX with a two-fold increase in basal ALP activity. While E2 or PTH+E2 had no effect on OP cells, both treatments inhibited ALP activity in CON cells (p<0.05). OP and CON cells grown in DEX also expressed PTH-stimulated adenylate cyclase (AC) activities higher than those of (-DEX) cells. OP+DEX cells, however, exhibited activities which were 8-fold higher than those of CON+DEX cells (p<0.001). In OP+DEX cells, E2 stimulated basal AC activity (p<0.05) but did not affect PTH-stimulated activity. In contrast, in CON+DEX cells, E2 had no effect on basal activity but inhibited PTH-stimulated AC activity (p<0.001). Osteocalcin production was 4-fold lower in OP+DEX cells compared to OP-DEX and CON cells (p<0.05) while osteocalcin mRNA levels were significantly lower in OP+DEX and CON+/-DEX cells compared to OP-DEX cells (p<0.05). E2 did not affect osteocalcin protein or mRNA levels in either OP or CON cells. No differences in mRNA levels were found for estrogen receptor-alpha (ER-a) in OP+/-DEX cells whereas these levels were significantly higher in CON+DEX compared to CON-DEX cells (p<0.05). These results indicate that DEX amplified the differences between OP and CON cells and confirm the presence of intrinsic osteoblastic abnormalities in patients with osteoporosis that persist in culture.

Cytotoxic T lymphocyte-associated antigen-4 inhibits integrin-mediated stimulation.

The negative role exerted by cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in the regulation of T-cell activity, as induced by T-cell receptor (TCR)/CD3 and CD28 costimulation, has been widely described. In the present work we investigated the role of CTLA-4 in the control of cell activation, as induced by costimulation of the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) in murine CD4+ T cells. Results show that CTLA-4 engagement inhibits interleukin-2 (IL-2) production, not only when induced by CD3/CD28 costimulation, but also when CD4+ T cells are costimulated by anti-CD3 and anti-LFA-1 monoclonal antibodies (mAbs). LFA-1 has been described to induce Ca2+ mobilization also in the absence of TCR engagement. Moreover, we found that CTLA-4 engagement negatively affects Ca2+ mobilization and NF-AT activation, as induced by LFA-1 engagement alone. PLCgamma1 phosphorylation was also dampened within minutes after CTLA-4 engagement. Altogether these data indicate that through the control of signals induced by different receptors, CTLA-4 could be a global attenuator of T-cell activation.

NADPH Oxidase-derived Reactive Oxygen Intermediates Regulate Tyrosine Phosphorylation in Human Neutrophils

Activation of the NADPH-oxidase in neutrophils results in the production of reactive oxygen intermediates (ROI) including superoxide (O2-) and hydrogen peroxide (H2O2) which have traditionally been viewed as functioning in host defense. Recent attention has been directed at a physiologic role for ROI as intracellular signaling molecules. Phosphorylation of proteins on tyrosine residues has been implicated in the control of cell activation, and the tyrosine kinase ltk in lymphocytes has been shown to be regulated by oxidants. As neutrophils produce prodigious quantities of ROI, we hypothesized that these compounds function in a physiological manner in the regulation of tyrosine phosphorylation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immuno-blotting with antiphosphotyrosine antibodies were used to assess tyrosine phosphorylation in human neutrophils. Exposure of neutrophils to diamide, a thiol-oxidizing agent, resulted in marked tyrosine phosphorylation of several proteins suggesting the regulation of this pathway by oxidant-sensitive enzymes. The following observations suggested that ROI produced by the NADPH-oxidase regulate tyrosine phosphorylation in a physiological manner: (1) direct activation of the oxidase by stimulation of G-proteins in electropermeabilized neutrophils by GTPγS or fluoroaluminate (AlF4-) resulted in marked tyrosine phosphorylation of bands of Mr = 30, 42, 60, and 90 in a time and concentration-dependent manner. Importantly, the latter effect was observed only when the cells were supplied with exogenous NADPH; (2) in the presence of NADPH but when the function of the oxidase was inhibited by diphenyleneiodonium, GTPγS-induced tyrosine phosphorylation was minimal; and (3) neutrophils from a patient with X-linked (X91°) chronic granulomatous disease (which are unable to produce ROI) did not demonstrate enhancement of GTPγS-induced tyrosine phosphorylation in the presence of NADPH. Using an in vitro gel renaturation assay with myelin basic protein as the substrate, we observed stimulation of kinase activity of a 42-kd band in cells treated with diamide suggesting that MAP kinase activity could be modulated by oxidants.