搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Immune tolerance fails in autoimmune polyendocrine syndrome type 1 (APS-1) because of AIRE mutations. We have used single cell transcriptomics to characterize regulatory T cells (Tregs) sorted directly from blood and from in vitro expanded Tregs in APS-1 patients compared to healthy controls. We revealed only CD52 and LTB (down) and TXNIP (up) as consistently differentially expressed genes in the datasets. There were furthermore no large differences of the TCR-repertoire of expanded Tregs between the cohorts,but unique patients showed a more restricted use of specific clonotypes. We also found that in vitro expanded Tregs from APS-1 patients had similar suppressive capacity as controls in co-culture assays,despite expanding faster and having more exhausted cells. Our results suggest that APS-1 patients do not have intrinsic defects in their Treg functionality,and that their Tregs can be expanded ex vivo for potential therapeutic applications. Subject areas: Health sciences,Immunology,Components of the immune system,Proteomics,Transcriptomics View Publication

Acute myeloid leukaemia (AML) is a disease of the haematopoietic stem cells(HSCs) that is characterised by the uncontrolled proliferation and impaired differentiation of normal haematopoietic stem/progenitor cells. Several pathways that control the proliferation and differentiation of HSCs are impaired in AML. Activation of the Wnt/beta-catenin signalling pathway has been shown in AML and beta-catenin,which is thought to be the key element of this pathway,has been frequently highlighted. The present study was designed to determine beta-catenin expression levels and beta-catenin-related genes in AML. In this study,beta-catenin gene expression levels were determined in 19 AML patients and 3 controls by qRT-PCR. Transcriptome analysis was performed on AML grouped according to beta-catenin expression levels. Differentially expressed genes(DEGs) were investigated in detail using the Database for Annotation Visualisation and Integrated Discovery(DAVID),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),STRING online tools. The transcriptome profiles of our AML samples showed different molecular signature profiles according to their beta-catenin levels(high-low). A total of 20 genes have been identified as hub genes. Among these,TTK,HJURP,KIF14,BTF3,RPL17 and RSL1D1 were found to be associated with beta-catenin and poor survival in AML. Furthermore,for the first time in our study,the ELOV6 gene,which is the most highly up-regulated gene in human AML samples,was correlated with a poor prognosis via high beta-catenin levels. It is suggested that the identification of beta-catenin-related gene profiles in AML may help to select new therapeutic targets for the treatment of AML. View Publication

BACKGROUND Adoptive cell therapy (ACT) using genetically modified T cells has evolved into a promising treatment option for patients with cancer. However,even for the best-studied and clinically validated CD19-targeted chimeric antigen receptor (CAR) T-cell therapy,many patients face the challenge of lack of response or occurrence of relapse. There is increasing need to improve the efficacy of ACT so that durable,curative outcomes can be achieved in a broad patient population. METHODS Here,we investigated the impact of indomethacin (indo),a non-steroidal anti-inflammatory drug (NSAID),on the efficacy of ACT in multiple preclinical models. Mice with established B-cell lymphoma received various combinations of preconditioning chemotherapy,infusion of suboptimal dose of tumor-reactive T cells,and indo administration. Donor T cells used in the ACT models included CD4+ T cells expressing a tumor-specific T cell receptor (TCR) and T cells engineered to express CD19CAR. Mice were monitored for tumor growth and survival. The effects of indo on donor T cell phenotype and function were evaluated. The molecular mechanisms by which indo may influence the outcome of ACT were investigated. RESULTS ACT coupled with indo administration led to improved tumor growth control and prolonged mouse survival. Indo did not affect the activation status and tumor infiltration of the donor T cells. Moreover,the beneficial effect of indo in ACT did not rely on its inhibitory effect on the immunosuppressive cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis. Instead,indo-induced oxidative stress boosted the expression of death receptor 5 (DR5) in tumor cells,rendering them susceptible to donor T cells expressing TNF-related apoptosis-inducing ligand (TRAIL). Furthermore,the ACT-potentiating effect of indo was diminished against DR5-deficient tumors,but was amplified by donor T cells engineered to overexpress TRAIL. CONCLUSION Our results demonstrate that the pro-oxidative property of indo can be exploited to enhance death receptor signaling in cancer cells,providing rationale for combining indo with genetically modified T cells to intensify tumor cell killing through the TRAIL-DR5 axis. These findings implicate indo administration,and potentially similar use of other NSAIDs,as a readily applicable and cost-effective approach to augment the efficacy of ACT. View Publication

OBJECTIVES We sought to develop medium throughput standard operating procedures for screening cryopreserved human peripheral blood mononuclear cells (PBMCs) for CD4+ and CD8+ T cell responses to potential autoantigens. METHODS Dendritic cells were loaded with a peptide cocktail from ubiquitous viruses or full-length viral protein antigens and cocultured with autologous T cells. We measured expression of surface activation markers on T cells by flow cytometry and cytometry by time of flight 24-72 h later. We tested responses among T cells freshly isolated from healthy control PBMCs,cryopreserved T cells,and T cells derived from a variety of T cell expansion protocols. We also compared the transcriptional profile of CD8+ T cells rested with interleukin (IL)7 for 48 h after 1) initial thawing,2) expansion,and 3) secondary cryopreservation/thawing of expanded cells. To generate competent antigen presenting cells from PBMCs,we promoted differentiation of PBMCs into dendritic cells with granulocyte macrophage colony stimulating factor and IL-4. RESULTS We observed robust dendritic cell differentiation from human PBMCs treated with 50 ng/mL GM-CSF and 20 ng/mL IL-4 in as little as 3 days. Dendritic cell purity was substantially increased by magnetically enriching for CD14+ monocytes prior to differentiation. We also measured antigen-dependent T cell activation in DC-T cell cocultures. However,polyclonal expansion of T cells with anti-CD3/antiCD28 abolished antigen-dependent upregulation of CD69 in our assay despite minimal transcriptional differences between rested CD8+ T cells before and after expansion. Furthermore,resting these expanded T cells in IL-2,IL-7 or IL-15 did not restore the antigen dependent responses. In contrast,T cells that were initially expanded with IL-2 + IL-7 rather than plate bound anti-CD3 + anti-CD28 retained responsiveness to antigen stimulation and these responses strongly correlated with responses measured at initial thawing. SIGNIFICANCE While screening techniques for potential pathological autoantibodies have come a long way,comparable full-length protein target assays for screening patient T cells at medium throughput are noticeably lacking due to technical hurdles. Here we advance techniques that should have broad applicability to translational studies investigating cell mediated immunity in infectious or autoimmune diseases. Future studies are aimed at investigating possible CD8+ T cell autoantigens in MS and other CNS autoimmune diseases. View Publication

Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function,DYSF exon 44,founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing,rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation,hEx44mut,recapitulates the patients’ phenotype and an identical re-framing outcome in primary muscle stem cells. Finally,gene-edited murine primary muscle stem-cells are able to regenerate muscle and rescue dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility,not only in treating dysferlin deficiency syndromes,but also perhaps other forms of muscular dystrophy. Dysferlin-deficient muscular dystrophy is a devastating and untreatable disease. Using Cas9,the authors restored dysferlin in muscle stem cells from patients ex vivo and show proof-of-concept for autologous cell replacement therapies in a new humanized mouse model. View Publication

In this study,we explore the therapeutic feasibility of globotriaosylceramide (Gb3) synthase (A4GALT)-specific siRNA-loaded polyhistidine (pHis)-incorporated lipid nanoparticles (HLNPs) for Fabry disease (FD). HLNPs were developed to deliver siRNAs targeting A4GALT using a microfluidic device,with pHis aiding in endosome escape. The therapy was tested on GLA-knockout human-induced pluripotent-stem-cell-derived endothelial cells (GLA-KO-hiPSC-ECs) and podocytes (GLA-KO-hiPSC-PCs). GLA-KO-hiPSCs-ECs or -PCs,upon differentiation,were treated with A4GALT-siRNA-HLNP. Successful intracellular uptake of A4GALT-siRNA-HLNP was confirmed through fluorescence and electron microscopy in both cell types. A4GALT-siRNA-HLNP treatment confirmed both cell types’ stability at 5 ?g/mL. Increased Gb3 deposition and zebra body formation were detected in both cell types,but A4GALT-siRNA-HLNP treatment attenuated these FD phenotypes,demonstrating reduced expression of A4GALT through western blot analysis. RNA sequencing analysis revealed that the expression of transcripts associated with FD was restored by A4GALT-siRNA-HLNP treatment in GLA-KO-hiPSCs-ECs,whereas in GLA-KO-hiPSCs-PCs,this effect was relatively less pronounced. Suppression of A4GALT via siRNA/HLNP treatment significantly rescued FD phenotypes especially in EC,presenting a novel therapeutic approach for FD. Graphical abstract This study highlights the therapeutic potential of A4GALT-siRNA delivered via HLNPs for Fabry disease (FD). In GLA-KO-hiPSC-derived endothelial cells and podocytes,treatment reduced Gb3 accumulation,restored transcriptomic changes,and mitigated FD phenotypes,with stronger effects in endothelial cells,supporting its promise as a novel FD therapy. View Publication

ABSTRACTThe gut microbiota and the immune system are closely connected,influencing early?life brain development. Brain?derived neurotrophic factor (BDNF),crucial for neuronal development,has been demonstrated to be produced by certain immune cells. However,the modulation of BDNF during bacterial antigen and metabolite challenge remains elusive. We investigate the effects of bacterial?derived antigens and metabolites on BDNF secretion in human PBMCs. Although BDNF levels were altered during stimulation,a specific cellular origin of BDNF within PBMCs was indeterminate. Positive magnetic separation of monocytes eliminated both the stimulant?induced BDNF secretion and reduced monocyte?platelet aggregates. Conversely,elevated platelet counts significantly increased BDNF levels,indicating that platelets,when interacting with monocytes and exposed to bacterial antigens,are likely the dominant source of BDNF in PBMC cultures. As previously described,platelets are a crucial source of circulating peripheral blood BDNF. Our findings emphasize the importance of the interplay between immune?blood cell complexes during microbial stimulation in regulating BDNF levels. This highlights the necessity of investigating such interactions to better understand the early?life gut?brain axis. Bacterial antigens primarily induce BDNF release from platelets interacting with monocytes in PBMCs. This interplay underscores how immune?blood cell complexes shape BDNF levels which may impact early human development. View Publication

Heat shock protein 90 (Hsp90) is a molecular chaperone important for maintaining protein homeostasis (proteostasis) in the cell. Hsp90 inhibitors are being explored as cancer therapeutics because of their ability to disrupt proteostasis. Inhibiting Hsp90 increases surface density of the immunological receptor Major Histocompatibility Complex 1 (MHC1). Here we show that this increase occurs across multiple cancer cell lines and with both cytosol-specific and pan-Hsp90 inhibitors. We demonstrate that Hsp90 inhibition also alters surface expression of both IFNGR and PD-L1,two additional immunological receptors that play a significant role in anti-tumour or anti-immune activity in the tumour microenvironment. Hsp90 also negatively regulates IFN-γ activity in cancer cells,suggesting it has a unique role in mediating the immune system’s response to cancer. Our data suggests a strong link between Hsp90 activity and the pathways that govern anti-tumour immunity. This highlights the potential for the use of an Hsp90 inhibitor in combination with another currently available cancer treatment,immune checkpoint blockade therapy,which works to prevent immune evasion of cancer cells. Combination checkpoint inhibitor therapy and the use of an Hsp90 inhibitor may potentiate the therapeutic benefits of both treatments and improve prognosis for cancer patients. View Publication

Engineered T cells equipped with a chimeric antigen receptor (CAR) have shown tremendous clinical success,but tumor-mediated stimulation of T cell inhibitory receptors leads to exhaustion,hampering durable remission in patients. Mitigation of this effect via checkpoint inhibition or genome editing to knockout the genes encoding for these receptors has shown promise. Yet,the side effects of these procedures require better alternatives. Targeted epigenome editing offers a potent strategy to alter gene expression without DNA modifications. Its hit-and-run mechanism enables durable,multiplexed modulation of gene expression with greater safety. Here,we describe multiplexed epigenome editing inactivation of two critical-exhaustion-related genes,PDCD1 and LAG3,both in primary human T cells and in prostate-cancer-specific CAR T cells. Epigenetically modified CAR T cells are indistinguishable from parental cells across a range of functional assays. Although the model does not fully mimic T cell exhaustion,limiting functional assessment,gene silencing remains durable across multiple divisions and repeated CAR stimulations. Furthermore,transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is effective and safe for multiplexed gene inhibition and holds potential in engineering CAR T cells with enhanced and customizable features. Graphical abstract Epigenome editing is used to engineer CAR T cells targeting prostate cancer by stably silencing the PDCD1 and LAG3 genes,which encode key inhibitory checkpoint receptors. This DNA break-free approach enhances safety by avoiding genomic damage and holds promise as a next-generation strategy for safer,more durable cancer immunotherapy. View Publication

Francisella tularensis (Ft),a Gram-negative bacterium that causes tularemia,possesses a non-inflammatory atypical LPS (LPSFt) that is highly immunogenic through unknown mechanism. We previously showed that immunization with LPSFt,a type 2 T-independent (TI) antigen,elicits protective LPSFt-specific IgM (IgMFt) and IgG3Ft by B1 cells in a mechanism dependent on the IL-5 produced by innate lymphoid cells type 2 (ILC2). Here,we examined the role of complement in the B1 cells’ response against LPSFt. C3-/-,C1q-/- and C4-/- mice immunized with LPSFt failed to produce IgMFt and IgG3Ft. In contrast,the response of Cfb-/- and Mbl1/Mbl2-/- mice was comparable to that of WT mice. Thus,activation of the classical complement cascade,but not the alternative or the Mannose Binding Lectin pathway,is required for activation of B1 cells and production of LPSFt-specific antibodies. Complement activation generates the C3d fragment,which opsonizes antigens for recognition by complement receptor-2 (CR2),and the C3a and C5a anaphylatoxins. Our results show that C3d opsonized LPSFt and that the response to immunization was dependent on CR2 expression by B1 cells. Importantly,the response to LPSFt immunization was also drastically decreased in C3ar1-/-,but not in C5ar1-/- mice. C3a induced IL-5 in ILC2,which supported B1 cells activation. Decreased antibody production in C3ar1-/- and Cr2-/- mice correlated with increased susceptibility to tularemia. Together,these results demonstrate that the high immunogenicity of LPSFt depends on two effector mechanisms triggered by activation of the classical complement pathway: 1) tagging of LPSFt with C3d fragment,leading to its interaction with CR2 expressed by B1 cells; 2) production of the anaphylatoxin C3a that stimulated IL-5 secretion by ILC2. Our study increases our understanding of the B1 cells’ response to TI-2 antigens and identifies two complement effector mechanisms that can be harnessed for therapeutic interventions. Author summaryThe lipopolysaccharide (LPS) of the bacterium Francisella tularensis strongly stimulates B cells for antibody production independently of T cell help through unknown mechanism. In the present study we examined the role of the complement cascade in this process. We found that production of antibodies against this LPS depends on activation of the classical complement pathway but not the MBL-dependent lectin or the alternative pathways. Following complement activation,LPS became tagged with the C3d complement fragment leading to its interaction with the complement receptor CR2 expressed by B cells. Complement activation also resulted in production of the anaphylatoxin C3a that was required for B cells activation,possibly through induction of IL-5 by innate lymphoid cells 2. Our study increases our understanding of the B cells’ response to T-independent antigens and identifies two complement effector mechanisms that can be harnessed for therapeutic interventions. View Publication

Human pluripotent stem cells including induced pluripotent stem cells (iPSCs) and embryonic stem cells hold great promise for cell-based therapies,but safety concerns that complicate consideration for routine clinical use remain. Installing a safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy we precisely installed the system into a genomic safe harbor the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1$\alpha$ promoter is silenced in iPSCs and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However the CAG promoter induces strong and stable iCASP9 expression in iPSCs and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives including MSCs and chondrocytes in vitro. Furthermore iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies." View Publication

The airway epithelium contains ionocytes,a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR),a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high ({\~{}}3{\%}) ionocyte abundance in ex vivo nasal samples,with no difference between CF and control individuals. In bronchi,ionocytes instead appeared very rarely as previously reported,thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture,which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure,we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions,thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern. View Publication