搜索结果: 'gmp t cell activator'

Regulatory T cells (Treg) play an important role in regulating immune homeostasis in health and disease. Traditionally their suppressive function has been assayed by mixing purified cell populations,which does not provide an accurate picture of a physiologically relevant response. To overcome this limitation,we here develop ‘single cell suppression profiling of human Tregs’ (scSPOT). scSPOT uses a 52-marker CyTOF panel,a cell division detection algorithm,and a whole PBMC system to assess the effect of Tregs on all other cell types simultaneously. In this head-to-head comparison,we find Tregs having the clearest suppressive effects on effector memory CD8 T cells through partial division arrest,cell cycle inhibition,and effector molecule downregulation. Additionally,scSPOT identifies a Treg phenotypic split previously observed in viral infection and propose modes of action by the FDA-approved drugs Ipilimumab and Tazemetostat. scSPOT is thus scalable,robust,widely applicable,and may be used to better understand Treg immunobiology and screen for therapeutic compounds. Traditional regulatory T cell (Tregs) assays utilize mixture of purified cell population. Here the authors develop a ‘single cell suppression profiling of human Tregs’ (scSPOT) with 52-marker CyTOF panel,a cell division detection algorithm,and a whole PBMC system to assess Treg suppressive function on all cell types simultaneously. View Publication

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here,we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits,both purified naive CD8+ T cells and purified naive CD4+ T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period,most of the cells died,but some survived in a quiescent state for a month. The survivors had small round cell bodies,expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia,if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats. View Publication

Pathobionts have evolved many strategies to coexist with the host,but how immune evasion mechanisms contribute to the difficulty of developing vaccines against pathobionts is unclear. Meanwhile,Staphylococcus aureus (SA) has resisted human vaccine development to date. Here we show that prior SA exposure induces non-protective CD4+ T cell imprints,leading to the blunting of protective IsdB vaccine responses. Mechanistically,these SA-experienced CD4+ T cells express IL-10,which is further amplified by vaccination and impedes vaccine protection by binding with IL-10Rα on CD4+ T cell and inhibit IL-17A production. IL-10 also mediates cross-suppression of IsdB and sdrE multi-antigen vaccine. By contrast,the inefficiency of SA IsdB,IsdA and MntC vaccines can be overcome by co-treatment with adjuvants that promote IL-17A and IFN-γ responses. We thus propose that IL-10 secreting,SA-experienced CD4+ T cell imprints represent a staphylococcal immune escaping mechanism that needs to be taken into consideration for future vaccine development. Mechanisms of inefficient vaccine protection against pathobionts such as S. aureus (SA) are still unclear. Here the authors show that prior SA exposure induces non-protective CD4+ T cells,which impair IsdB vaccine protection by IL-10 secretion and IL-17A suppression,whereas IL-17A promoting adjuvant CAF01 overcomes this dilemma. View Publication

Upon immunogenic challenge,lymph nodes become mechanically stiff as immune cells activate and proliferate within their encapsulated environments,and with resolution,they reestablish a soft baseline state. Here we show that sensing these mechanical changes in the microenvironment requires the mechanosensor YAP. YAP is induced upon activation and suppresses metabolic reprogramming of effector T cells. Unlike in other cell types in which YAP promotes proliferation,YAP in T cells suppresses proliferation in a stiffness-dependent manner by directly restricting the translocation of NFAT1 into the nucleus. YAP slows T cell responses in systemic viral infections and retards effector T cells in autoimmune diabetes. Our work reveals a paradigm whereby tissue mechanics fine-tune adaptive immune responses in health and disease. View Publication

Most cancer vaccines target peptide antigens,necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore,tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells,but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding,enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably,this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary,highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations. View Publication

We used primary peripheral blood T cells,a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously,to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly,T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that,in the face of chronic nicotine exposure,selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases. View Publication

T cell homeostasis is crucial for maintaining an efficient and balanced T cell immunity. The interaction between TCR and self peptide (sp) MHC ligands is known to be the key driving force in this process,and it is believed to be functionally and mechanistically different from that initiated by the antigenic TCR stimulation. Yet,very little is known about the downstream signaling events triggered by this TCR-spMHC interaction and how they differ from those triggered by antigenic TCR stimulation. In this study,we show that T cell conditional ablation of MEKK3,a Ser/Thr kinase in the MAPK cascade,causes a significant reduction in peripheral T cell numbers in the conditional knockout mice,but does not perturb thymic T cell development and maturation. Using an adoptive mixed transfer method,we show that MEKK3-deficient T cells are severely impaired in lymphopenia-induced cell proliferation and survival. Interestingly,the Ag-induced T cell proliferation proceeds normally in the absence of MEKK3. Finally,we found that the activity of ERK1/2,but not p38 MAPK,was attenuated during the lymphopenia-driven response in MEKK3-deficient T cells. Together,these data suggest that MEKK3 may play a crucial selective role for spMHC-mediated T cell homeostasis. View Publication

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse,we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding,at the molecular level,how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here,we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally,we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization,as well as T cell migration in vitro. Altogether,our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility. View Publication

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However,further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-$\gamma$. In addition,we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-$\beta$ receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells,LTBR induced profound transcriptional and epigenomic remodelling,leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-$\kappa$B pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and ?? T cells,highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes. View Publication

Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections,we investigated the function of the adhesion molecule,P-selectin glycoprotein ligand-1 (PSGL-1),that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably,this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically,PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1,leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs,PSGL-1 deficiency led to PD-1 downregulation,improved T cell responses,and tumor control. Thus,PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment. View Publication

CD4 T cells from HIV-1 infected patients die at excessive rates compared to those from uninfected patients,causing immunodeficiency. We previously identified a dominant negative ligand that antagonizes the TRAIL-dependent pathway of cell death,which we called TRAILshort. Because the TRAIL pathway has been implicated in CD4 T cell death occurring during HIV-1 infection,we used short hairpin RNA knockdown,CRISPR deletion,or Abs specific for TRAILshort to determine the effect of inhibiting TRAILshort on the outcome of experimental acute HIV infection in vitro. Strikingly,all three approaches to TRAILshort deletion/inhibition enhanced HIV-induced death of both infected and uninfected human CD4 T cells. Thus,TRAILshort impacts T cell dynamics during HIV infection,and inhibiting TRAILshort causes more HIV-infected and uninfected bystander cells to die. TRAILshort is,therefore,a host-derived,host-adaptive mechanism to limit the effects of TRAIL-induced cell death. Further studies on the effects of TRAILshort in other disease states are warranted. View Publication

NK cell transcript 4 (NK4),now denoted as IL-32,was originally identified as a transcript whose expression was increased in activated NK cells. It has been very recently demonstrated that NK4 is secreted from several cells upon the stimulation of some inflammatory cytokines such as IL-18,IL-1beta,IFN-gamma and IL-12. Furthermore,NK4 induces production of tumor necrosis factor,macrophage inflammatory protein (MIP)-2 and IL-8 in monocytic cell lines,indicating that this factor would be involved in the inflammatory responses. Based on these findings,NK4 was renamed IL-32. However,the biological activities of IL-32 on other cell types remained undetermined. Furthermore,it was still argued whether IL-32 acts on cells from outside or inside the cells. In this article,we first report that expression of IL-32 was up-regulated in activated T cells and NK cells,and that IL-32beta was the predominantly expressed isoform in activated T cells. IL-32 was specifically expressed in T cells undergoing apoptosis and enforced expression of IL-32-induced apoptosis,whereas its down-regulation rescued the cells from apoptosis in HeLa cells. IL-32 existing in the supernatant would be derived from the cytoplasm of apoptotic cells. These results strongly indicated that IL-32 would be involved in activation-induced cell death in T cells,probably via its intracellular actions. Our present findings expand our understanding of the biological function of IL-32 and argue that IL-32 may act on cells,not only from the outside but also from the inside. View Publication