Defining the mechanism of action in a novel class of anti-PD-1 antibodies

Background. Despite the relative success of anti-PD-1 antibodies over the years in cancer immunotherapy, the mechanism of action beyond blockade of the ligand binding site remains inadequately defined. Given that anti-PD-1 therapy is effective in only a fraction of cancer patients, it is imperative to elucidate the mechanisms imparted by anti-PD-1 antibodies and how they are involved in restoring activity to functionally exhausted PD-1+ memory T cells. Hypothesis. The two hypotheses investigated are: 1) The novel non-blocking anti-PD-1 antibody discovered in our lab differs in its mechanism of action from the traditional blocking PD-1 antibodies, 2) Anti-PD-1 mediated downregulation contributes to the relief of T cell exhaustion and occurs through an antibody-Fc interaction with FcγR expressing cells. Objectives. The research plans are: 1) to develop an experimental strategy to evaluate the functional activity in primary, memory T cells responding to the anti-PD-1 therapies utilizing coinhibitory conditions with PD-L1 and 2) to identify the cell populations and associated determinants for anti-PD-1 mediated downregulation on primary, memory T cells. Experimental strategy. 1) We used PBMCs isolated from chronically infected HIV+ donors with high expression of PD-1 to measure the activation of key mediators of the T cell activation pathway. We were able to evaluate the restoration of phosphoprotein activity and calcium signaling to these functionally exhausted primary T cells at early timepoints when stimulated by CD3/CD28/PD-L1 and treated with anti-PD-1. 2) We isolated different fractions of the PBMC composition to determine if any effector cells were involved in PD-1 downregulation. Furthermore, we sought to establish a contact-based dependency test to determine which receptor was necessary for the anti-PD-1 downregulation effect. Results. 1) We demonstrated that both blocking and non-blocking antibodies were able to restore T cell signaling but the non-blocking antibody restored significantly more Akt signaling and the combination of the two anti-PD-1 antibodies enhanced the restoration of Ca2+ signaling dramatically. 2) Anti-PD-1 mediated downregulation was observed only in the presence of monocytes and CD64 was found to be the FcγR with the highest impact for this effect. Conclusions. 1) The non-blocking PD-1 antibody was shown to be effective in restoring T cell functionality and appeared to preferentially restore signaling through the CD28 associated pathway while the blocking PD-1 antibody exerted its effect over the TCR pathway. 2) The requirement for CD64 expressing monocytes was shown for the antibody-mediated downregulation of PD-1 on memory T cells. -- Contexte: Malgré le succès relatif des anticorps anti-PD-1 au fil des années dans l'immunothérapie anticancéreuse, le mécanisme d'action au-delà du blocage du site de liaison du ligand reste mal défini. Étant donné que le traitement anti-PD-1 n'est efficace que chez une fraction des patients cancéreux, il est impératif d'élucider les mécanismes conférés par les anticorps anti-PD-1 et comment ils sont impliqués dans la restauration de l'activité de la mémoire PD-1+ fonctionnellement épuisée T cellules. Hypothèse: Les deux hypothèsesétudiées sont: 1) Le nouvel anticorps anti-PD-1 non bloquant découvert dans notre laboratoire diffère dans son mécanisme d'action des anticorps bloquants traditionnels PD-1, 2) La régulation négative médiée par anti-PD-1 contribue à la soulagement de l'épuisement des cellules T et se produit par le biais d'une interaction anticorps-Fc avec les cellules exprimant FcγR. Objectifs: Les plans de recherche sont les suivants: 1) développer une stratégie expérimentale pour évaluer l'activité fonctionnelle des lymphocytes T mémoires primaires répondant aux thérapies anti-PD-1 utilisant des conditions co-inhibitrices avec PD-L1 et 2) pour identifier les populations cellulaireset les déterminants associés pour la régulation négative à médiation anti- PD-1 sur les lymphocytes T primaires à mémoire. Stratégie expérimentale: 1) Nous avons utilisé des PBMC isolées de donneurs VIH + infectés de manière chronique avec une forte expression de PD-1 pour mesurer l'activation de médiateurs clés de la voie d'activation des lymphocytes T. Nous avons pu évaluer la restauration de l'activité des phosphoprotéines et la signalisation du calcium vers ces lymphocytes T primaires fonctionnellement épuisés à des moments précoces lorsqu'ils sont stimulés par CD3 / CD28 / PD-L1 et traités avec de l'anti-PD-1. 2) Nous avons isolé différentes fractions de la composition de PBMC pour déterminer si des cellules effectrices étaient impliquées dans la régulation négative de PD-1. En outre, nous avons cherché à établir un test de dépendance basé sur le contact pour déterminer quel récepteur était nécessaire pour l'effet de régulation négative anti-PD-1. Résultats: 1) Nous avons démontré que les anticorps bloquants et non bloquants étaient capables de restaurer la signalisation des lymphocytes T, mais l'anticorps non bloquant a restauré significativement plus de signalisation Akt et la combinaison des deux anticorps anti- PD-1 a considérablement amélioré la restauration de la signalisation Ca 2+. 2) une régulation négative à médiation anti-PD-1 a été observée uniquement en présence de monocytes et CD64 s'est avéré être le FcyR avec l'impact le plus élevé pour cet effet. Conclusion: 1) L'anticorps PD-1 non bloquant s'est avéré efficace pour restaurer la fonctionnalité des lymphocytes T et semblait restaurer préférentiellement la signalisation via la voie associée au CD28 tandis que l'anticorps bloquant PD-1 exerçait son effet sur la voie TCR. 2) L'exigence de monocytes exprimant CD64 a été montrée pour la régulation à la baisse médiée par les anticorps de PD-1 sur les cellules T mémoire

Automatic Intent-Based Secure Service Creation Through a Multilayer SDN Network Orchestration

Growing traffic demands and increasing security awareness are driving the need for secure services. Current solutions require manual configuration and deployment based on the customer's requirements. In this work, we present an architecture for an automatic intent-based provisioning of a secure service in a multilayer - IP, Ethernet, and optical - network while choosing the appropriate encryption layer using an open-source software-defined networking (SDN) orchestrator. The approach is experimentally evaluated in a testbed with commercial equipment. Results indicate that the processing impact of secure channel creation on a controller is negligible. As the time for setting up services over WDM varies between technologies, it needs to be taken into account in the decision-making process.Comment: Parts of the presented work has received funding from the European Commission within the H2020 Research and Innovation Programme, under grant agreeement n.645127, project ACIN

Anomaly detection through enhanced sentiment analysis on social media data

Agency for Science, Technology and Research (A*STAR

Efficient Mixing at low Reynolds numbers using polymer additives

Mixing in fluids is a rapidly developing field of fluid mechanics \cite{Sreen,Shr,War}, being an important industrial and environmental problem. The mixing of liquids at low Reynolds numbers is usually quite weak in simple flows, and it requires special devices to be efficient. Recently, the problem of mixing was solved analytically for a simple case of random flow, known as the Batchelor regime \cite{Bat,Kraich,Fal,Sig,Fouxon}. Here we demonstrate experimentally that very viscous liquids at low Reynolds number, $Re$. Here we show that very viscous liquids containing a small amount of high molecular weight polymers can be mixed quite efficiently at very low Reynolds numbers, for a simple flow in a curved channel. A polymer concentration of only 0.001% suffices. The presence of the polymers leads to an elastic instability \cite{LMS} and to irregular flow \cite{Ours}, with velocity spectra corresponding to the Batchelor regime \cite{Bat,Kraich,Fal,Sig,Fouxon}. Our detailed observations of the mixing in this regime enable us to confirm sevearl important theoretical predictions: the probability distributions of the concentration exhibit exponential tails \cite{Fal,Fouxon}, moments of the distribution decay exponentially along the flow \cite{Fouxon}, and the spatial correlation function of concentration decays logarithmically.Comment: 11 pages, 5 figure

Separable mechanisms drive local and global polarity establishment in the Caenorhabditis elegans intestinal epithelium

Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resilience to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue-specific protein depletion in the Caenorhabditis elegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but local polarity can arise in its absence, as small groups of cells eventually established polarized domains in PAR-3-depleted intestines in a HMR-1 (E-cadherin)dependent manner. Despite the role of PAR-3 in localizing PKC-3 to the apical surface, we additionally found that PAR-3 and PKC-3/ aPKC have distinct roles in the establishment and maintenance of local and global polarity. Taken together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo

Establishing criteria for human mesenchymal stem cell potency

This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age‐ and sex‐matched donors. Adherence to plastic was not indicative of potency, yet capacity for long‐term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high‐growth capacity or low‐growth capacity. Using this grouping strategy, high‐growth capacity MSCs were smaller in size, had greater colony‐forming efficiency, and had longer telomeres. Cell‐surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high‐growth capacity and low‐growth capacity MSCs, whereas STRO‐1 and platelet‐derived growth factor receptor alpha were preferentially expressed on high‐growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST‐1 and DERMO‐1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high‐growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low‐growth capacity MSCs when assessed for ectopic bone‐forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application

Effects of cultural tightness-looseness and social network density on expression of positive and negative emotions: A large-scale study of impression management by Facebook users

A*Star; Singapore Management Universit

Diffusion-Weighted MRI in Recurrent Wernicke's Encephalopathy: a Remarkable Cerebellar Lesion

We report unusual MRI findings (including those from diffusion-weighted imaging (DWI)) in a patient with recurrent Wernicke's encephalopathy with a remarkable cerebellar lesion. DWI showed high signal intensities in the superior portion of the cerebellar hemisphere and vermis area. After thiamine administration, clinical symptoms improved and the lesions with high signal intensities disappeared on follow-up DWI

Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival

Elastic turbulence in curvilinear flows of polymer solutions

Following our first report (A. Groisman and V. Steinberg, \sl Nature $\bf 405$, 53 (2000)) we present an extended account of experimental observations of elasticity induced turbulence in three different systems: a swirling flow between two plates, a Couette-Taylor (CT) flow between two cylinders, and a flow in a curvilinear channel (Dean flow). All three set-ups had high ratio of width of the region available for flow to radius of curvature of the streamlines. The experiments were carried out with dilute solutions of high molecular weight polyacrylamide in concentrated sugar syrups. High polymer relaxation time and solution viscosity ensured prevalence of non-linear elastic effects over inertial non-linearity, and development of purely elastic instabilities at low Reynolds number (Re) in all three flows. Above the elastic instability threshold, flows in all three systems exhibit features of developed turbulence. Those include: (i)randomly fluctuating fluid motion excited in a broad range of spatial and temporal scales; (ii) significant increase in the rates of momentum and mass transfer (compared to those expected for a steady flow with a smooth velocity profile). Phenomenology, driving mechanisms, and parameter dependence of the elastic turbulence are compared with those of the conventional high Re hydrodynamic turbulence in Newtonian fluids.Comment: 23 pages, 26 figure