29 research outputs found
Toward high-throughput engineering techniques for improving CAR intracellular signaling domains
Chimeric antigen receptors (CAR) are generated by linking extracellular antigen recognition domains with one or more intracellular signaling domains derived from the T-cell receptor complex or various co-stimulatory receptors. The choice and relative positioning of signaling domains help to determine chimeric antigen receptors T-cell activity and fate in vivo. While prior studies have focused on optimizing signaling power through combinatorial investigation of native intracellular signaling domains in modular fashion, few have investigated the prospect of sequence engineering within domains. Here, we sought to develop a novel in situ screening method that could permit deployment of directed evolution approaches to identify intracellular domain variants that drive selective induction of transcription factors. To accomplish this goal, we evaluated a screening approach based on the activation of a human NF-κB and NFAT reporter T-cell line for the isolation of mutations that directly impact T cell activation in vitro. As a proof-of-concept, a model library of chimeric antigen receptors signaling domain variants was constructed and used to demonstrate the ability to discern amongst chimeric antigen receptors containing different co-stimulatory domains. A rare, higher-signaling variant with frequency as low as 1 in 1000 could be identified in a high throughput setting. Collectively, this work highlights both prospects and limitations of novel mammalian display methods for chimeric antigen receptors signaling domain discovery and points to potential strategies for future chimeric antigen receptors development
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Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.
We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations
Immune-checkpoint Protein VISTA Critically Regulates the IL-23/IL-17 Inflammatory Axis
V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T cell activation when expressed on antigen-presenting cells. Vsir−/− mice developed loss of peripheral tolerance and multi-organ chronic inflammatory phenotypes. Vsir−/− CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Whether or not VISTA regulates innate immunity is unknown. Using a murine model of psoriasis induced by TLR7 agonist imiquimod (IMQ), we show that VISTA deficiency exacerbated psoriasiform inflammation. Enhanced TLR7 signaling in Vsir−/− dendritic cells (DCs) led to the hyper-activation of Erk1/2 and Jnk1/2, and augmented the production of IL-23. IL-23, in turn, promoted the expression of IL-17A in both TCRγδ+ T cells and CD4+ Th17 cells. Furthermore, VISTA regulates the peripheral homeostasis of CD27− γδ T cells and their activation upon TCR-mediated or cytokine-mediated stimulation. IL-17A-producing CD27− γδ T cells were expanded in the Vsir−/− mice and amplified the inflammatory cascade. In conclusion, this study has demonstrated that VISTA critically regulates the inflammatory responses mediated by DCs and IL-17-producing TCRγδ+ and CD4+ Th17 T cells following TLR7 stimulation. Our finding provides a rationale for therapeutically enhancing VISTA-mediated pathways to benefit the treatment of autoimmune and inflammatory disorders
The Wnt/Planar Cell Polarity Pathway Component Vangl2 Induces Synapse Formation through Direct Control of N-Cadherin
Although regulators of the Wnt/planar cell polarity (PCP) pathway are widely expressed in vertebrate nervous systems, their roles at synapses are unknown. Here, we show that Vangl2 is a postsynaptic factor crucial for synaptogenesis and that it coprecipitates with N-cadherin and PSD-95 from synapse-rich brain extracts. Vangl2 directly binds N-cadherin and enhances its internalization in a Rab5-dependent manner. This physical and functional interaction is suppressed by β-catenin, which binds the same intracellular region of N-cadherin as Vangl2. In hippocampal neurons expressing reduced Vangl2 levels, dendritic spine formation as well as synaptic marker clustering is significantly impaired. Furthermore, Prickle2, another postsynaptic PCP component, inhibits the N-cadherin-Vangl2 interaction and is required for normal spine formation. These results demonstrate direct control of classic cadherin by PCP factors; this control may play a central role in the precise formation and maturation of cell-cell adhesions at the synapse
Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses
IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca2+through the noncanonical STAT3 pathway
Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca2+. Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca2+-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role
Mst1 Directs Myosin IIa Partitioning of Low and Higher Affinity Integrins during T Cell Migration
<div><p>Chemokines promote T cell migration by transmitting signals that induce T cell polarization and integrin activation and adhesion. Mst1 kinase is a key signal mediator required for both of these processes; however, its molecular mechanism remains unclear. Here, we present a mouse model in which Mst1 function is disrupted by a hypomorphic mutation. Microscopic analysis of <i>Mst1</i>-deficient CD4 T cells revealed a necessary role for Mst1 in controlling the localization and activity of Myosin IIa, a molecular motor that moves along actin filaments. Using affinity specific LFA-1 antibodies, we identified a requirement for Myosin IIa-dependent contraction in the precise spatial distribution of low and higher affinity LFA-1 on the membrane of migrating T cells. <i>Mst1</i> deficiency or Myosin inhibition resulted in multipolar cells, difficulties in uropod detachment and mis-localization of low affinity LFA-1. Thus, Mst1 regulates Myosin IIa dynamics to organize high and low affinity LFA-1 to the anterior and posterior membrane during T cell migration.</p></div