搜索结果: 'gmp t cell activator'

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases,including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs,most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets,may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes,a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern,we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39− CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly,β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary,introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

T-cell genome engineering holds great promise for cell-based therapies for cancer,HIV,primary immune deficiencies,and autoimmune diseases,but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types� View Publication

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach,however,is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here,we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT." View Publication

Halofuginone,a low molecular weight plant alkaloid,inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease,including scleroderma and graft-versus-host disease. In addition,halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-beta (TGF-beta),an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-kappaB activity,production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma),T cell apoptosis,chemotaxis,and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng halofuginone,P = 0.002). In addition,40 ng/ml halofuginone inhibited secretion of TNF-alpha,IFN-gamma,interleukin (IL)-4,IL-13,and TGF-beta (P textless 0.005). Similarly,halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005,respectively). In contrast,T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice,indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo,making it an attractive immunomodulator and anti-inflammatory agent. View Publication

T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells,much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation,we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc,IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach,we identified textgreater200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism,and consistent with a connection between O-GlcNAc and RNA,inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall,our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization,to our knowledge,of the O-GlcNAc glycoproteome in human T cells. View Publication

Immune checkpoint blockade has revolutionized the field of oncology,inducing durable anti-tumour immunity in solid tumours. In patients with advanced prostate cancer,immunotherapy treatments have largely failed1-5. Androgen deprivation therapy is classically administered in these patients to inhibit tumour cell growth,and we postulated that this therapy also affects tumour-associated T cells. Here we demonstrate that androgen receptor (AR) blockade sensitizes tumour-bearing hosts to effective checkpoint blockade by directly enhancing CD8 T cell function. Inhibition of AR activity in CD8 T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy via increased IFN$\gamma$ expression. AR bound directly to Ifng and eviction of AR with a small molecule significantly increased cytokine production in CD8 T cells. Together,our findings establish that T cell intrinsic AR activity represses IFN$\gamma$ expression and represents a novel mechanism of immunotherapy resistance. View Publication

Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges,including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer,functional,and tunable alternative for gene silencing without the need for DNA editing. As a proof of concept for multiplex gene silencing,we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors,enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models. Silenced (S) M5CAR T cells retained antitumor functionality comparable to,and in some cases exceeding,that of KO cells. In vivo,S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs). Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells,with potential advantages in potency,persistence,metastasis prevention,and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks. View Publication

NLRC5 is a transcriptional regulator of MHC class I (MHCI),which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK-T-cell crosstalk,raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly,NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice,we show that NK cells surprisingly break tolerance even towards 'self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore,during chronic LCMV infection,the total CD8(+) T-cell population is severely decreased in these mice,a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets,having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions. View Publication

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore,we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that,in the absence of APCs,flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-gamma,IL-8,and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS,ligands for TLR3 and TLR4,respectively,was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover,among the memory T cells,CCR7- effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells,and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production. View Publication

Although CD31 expression on human thymocytes has been reported,a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. In this study,we provide a global picture of the evolution of CD31 expression from the CD34(+) hematopoietic precursor to the CD45RA(+) mature CD4(+) and CD8(+) single-positive (SP) T cells. Using nine-color flow cytometry,we show that CD31 is highly expressed on CD34(+) progenitors and stays high until the early double-positive stage (CD3(-)CD4(+)CD8α(+)β(-)). After β-selection,CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3(high)CD4(+)CD8(+) double-positive thymocytes. However,following positive selection,CD31 expression differs dramatically between CD4(+) and CD8(+) lineages: homogeneously high on CD8 SP but lower or negative on CD4 SP cells,including a subset of CD45RA(+)CD31(-) mature CD4(+) thymocytes. CD31 expression on TCRγδ thymocytes is very similar to that of CD4 SP cells. Remarkably,there is a substantial subset of semimature (CD45RA(-)) CD4 SP thymocytes that lack CD31 expression. Moreover,FOXP3(+) and ICOS(+) cells are overrepresented in this CD31(-) subpopulation. Despite this CD31(-)CD45RA(-) subpopulation,most egress-capable mature CD45RA(+) CD4 SP thymocytes express CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: β-selection,positive selection,and negative selection. Considering the ability of CD31 to modulate the TCR's activation threshold via the recruitment of tyrosine phosphatases,our results suggest a significant role for CD31 during T cell development. View Publication

The lymph node (LN) performs essential roles in immunosurveillance throughout the body. Developing in vitro models of this key tissue is of great importance to enhancing physiological relevance in immunoengineering. The LN consists of stromal populations and immune cells,which are highly organized and bathed in constant interstitial fluid flow (IFF). The stroma,notably the fibroblastic reticular cells (FRCs) and the lymphatic endothelial cells (LECs),play crucial roles in guiding T cell migration and are known to be sensitive to fluid flow. During inflammation,interstitial fluid flow rates drastically increase in the LN. It is unknown how these altered flow rates impact crosstalk and cell behavior in the LN,and most existing in vitro models focus on the interactions between T cells,B cells,and dendritic cells rather than with the stroma. To address this gap,we developed a human engineered model of the LN stroma consisting of FRC-laden hydrogel above a monolayer of LECs in a tissue culture insert with gravity-driven interstitial flow. We found that FRCs had enhanced coverage and proliferation in response to high flow rates,while LECs experienced decreased barrier integrity. We added CD4+ and CD8+ T cells and found that their egress was significantly decreased in the presence of interstitial flow,regardless of magnitude. Interestingly,3.0 μ m/s flow,but not 0.8 μ m/s flow,correlated with enhanced inflammatory cytokine secretion in the LN stroma. Overall,we demonstrate that interstitial flow is an essential consideration in the lymph node for modulating LN stroma morphology,T cell migration,and inflammation. View Publication