搜索结果: 'gmp t cell activator'

The interaction between the linker for activation of T cells (LAT) with PLC-gamma1 is important for TCR-mediated Ca(2+) signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells,implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect,we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wild type LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling,proliferation,and IL-2 production. Furthermore,the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3(+) natural Treg cells were present in these mice after deletion,they were unable to suppress the proliferation of conventional T cells. Our data indicate that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4(+)CD25(+) regulatory T cells. View Publication

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies,including acute myeloid leukemia (AML). Here,we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this,we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78,CD123,or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus,we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations. View Publication

Genetically modified T lymphocytes expressing chimeric antigen receptors (CARs) are becoming increasingly important in the treatment of hematologic malignancies and are also intensively being investigated for other diseases such as autoimmune disorders and HIV. Current CAR T cell therapies predominantly use viral transduction methods which,despite their efficacy,raise safety concerns related to genomic integration and potentially associated malignancies as well as labor- and cost-intensive manufacturing. Therefore,non-viral gene transfer methods,especially mRNA-based approaches,have attracted research interest due to their transient modification and enhanced safety profile. In this study,the optimization of CAR-mRNA for T cell applications is investigated,focusing on the impact of mRNA modifications,in vitro transcription protocols,and purification techniques on the translation efficiency and immunogenicity of mRNA. Furthermore,the refined CAR-mRNA was used to generate transient CAR T cells from acute myeloid leukemia patient samples,demonstrating efficacy in vitro and proof-of-concept for clinically relevant settings. These results highlight the potential of optimized mRNA to produce transient and safe CAR T cells. View Publication

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained,and the molecular programmes defining the autoimmune T cell state,are unknown. In type 1 diabetes,$\beta$-cell-specific CD8 T cells destroy insulin-producing $\beta$-cells. Here we followed the fate of $\beta$-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN),which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas,where they differentiate further and destroy $\beta$-cells. Whereas transplantation of as few as 20 ?»¿autoimmune progenitors induced type 1 diabetes,as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived,and stem-like ?»¿autoimmune progenitors must continuously seed the pancreas to sustain $\beta$-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes. View Publication

B cell-activating factor of the tumor necrosis factor (TNF) family,or BAFF,is mainly produced in monocytes and dendritic cells,and indispensable for proliferation,differentiation and survival of B cells. BAFF is a type II membrane-bound protein and the extracellular C-terminal fragment is released from the cells as soluble BAFF (sBAFF),which binds to specific receptors on B cells. Accumulating evidence suggests that BAFF plays an important role in the pathogenesis of autoimmune diseases,such as systemic lupus erythematosus (SLE). In this study,we developed a sensitive sandwich ELISA system to quantify the amount of sBAFF using our own mAb. Treatment of peripheral T cells of SLE patients with an anti-CD3 antibody triggered robust expression of BAFF and subsequent release of sBAFF from the cells. On the other hand,the stimulus induced only marginal elevation of sBAFF from normal T cells. These data indicate that BAFF is expressed in T cells upon stimulation at least under pathological conditions. Expression of BAFF was also largely induced in a human T cell line,Loucy (American Type Tissue Collection CRL-2629),in response to several stimuli,while other T cell lines so far examined produced the cytokine almost constitutively. These data suggest that Loucy recapitulates some of the characteristics of SLE T cells. Investigation of molecular and cellular mechanisms of production of BAFF in Loucy demonstrated that expression of BAFF was regulated through a signal transduction pathway which involves c-jun NH2-terminal kinase and p38,and that shedding of BAFF was catalyzed by a membrane-bound protease,furin. View Publication

Human pluripotent stem cells (PSCs),which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs),represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such,it is becoming increasingly important to develop methods for the large-scale banking of human PSC lines. There are several well-established methods for the propagation of human PSCs. The key to development of a good manufacturing practice (GMP) bank is to determine a manufacturing method that is amenable to large-scale production using materials that are fully documented. We have developed several banks of hESCs using animal feeder cells,animal-based matrices,or animal-free matrices. Protocols for growing hESCs on mouse embryonic fibroblasts (MEFs) are well established and are very helpful for producing research grade banks of cells. As most human ESCs cultured by research laboratories have been exposed to xenogeneic reagents,it is not imperative that all materials used in the production of a master cell bank be animal-free in origin. Nevertheless,as the field develops,it will no doubt become increasingly important to produce a bank of cells for clinical use without xenogeneic reagents,particularly nonhuman feeder cells which might harbor viruses with potential risk to human health or cell product integrity. Thus,even for cell lines previously exposed to xenogeneic reagents,it is important to minimize any subsequent exposure of the cell lines to additional adventitious agents. We have specifically described procedures for the growth of hESCs on Matrigel,an animal-matrix,and CELLstart,an animal-free matrix,and these can be used to produce hESCs as part of a clinical manufacturing process. View Publication

High-dose,posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However,the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide,a cyclophosphamide (Cy) analog,on CD4(+) T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve,potentially alloreactive conventional CD4(+) T cells with relative preservation of memory CD4(+)Foxp3(+) T cells. In particular,CD4(+)CD45RA(-)Foxp3(+hi) effector regulatory T cells (Tregs) recovered rapidly after alloBMT and,unexpectedly,were present at higher levels in patients with GVHD. CD4(+)Foxp3(+) T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH),the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4(+) T cells and sensitized Tregs to mafosfamide. Finally,removing Tregs from peripheral blood lymphocyte grafts obviated PTCy's GVHD-protective effect in a xenogeneic transplant model. Together,these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD. View Publication

Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies,the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner,making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN),complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2,with preclinical evidence to support their efficacy in T cell malignancies. The T cell receptor β-chain is expressed in two isoforms,TRBC1 and TRBC2,with clonally expanded mature T cell lymphomas expressing one of them exclusively,while healthy T cells randomly express either TRBC1 or TRBC2. Here authors show structure-based design of a TRBC2-specific antibody,and depletion of malignant T cells carrying TRBC1 or TRBC2 with CAR-T cells against the cognate receptor chain in murine models. View Publication

The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells,whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN,including pre-TFH cells,memory TFH cells,germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages,including multiple responses targeting internal influenza virus proteins,and found that each TFH cell state was attainable within a clonal lineage. Thus,human TFH cells form a durable and dynamic multitissue network. Schattgen et al. profiled the subsets and clonality of CD4+ TFH cells in the blood and lymph nodes of human volunteers who received two influenza vaccines 1 year apart to characterize their dynamics and clonal evolution over 2 years. View Publication

Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity,yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here,we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation,which revealed distinct gene networks individually regulated by FOXP3 and PRDM1,in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2,to our knowledge not previously implicated in Treg cell function,coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling,we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies. View Publication

CD8+ T cell exhaustion (Tex) limits immune control of cancer,but the underlying molecular drivers are unclear. In the present study,we identified the prostaglandin I2 (prostacyclin) receptor PTGIR as a cell-intrinsic regulator of T cell exhaustion. Transcriptomic profiling of terminally exhausted (Ttex) CD8+ T cells revealed increased activation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response pathway. Enhancing NRF2 activity (by conditional deletion of Kelch-like ECH-associated protein 1 (KEAP1)) boosts glutathione production in CD8+ T cells but accelerates terminal exhaustion. NRF2 upregulates PTGIR expression in CD8+ T cells. Silencing PTGIR expression enhances T cell effector function (that is,interferon-γ and granzyme production) and limits Ttex cell development in chronic infection and cancer models. Mechanistically,PTGIR signaling impairs T cell metabolism and cytokine production while inducing transcriptional features of Tex cells. These findings identify PTGIR as a NRF2-dependent immune checkpoint that regulates balance between effector and exhausted CD8+ T cell states. Targeting CD8+ T cell exhaustion is a strategy to enhance immune checkpoint inhibition and to fight cancer. Here the authors show a NRF2-dependent role for the prostaglandin I2 receptor PTGIR in controlling T cell exhaustion. View Publication

Mucosal-associated invariant T (MAIT) cells exhibit different characteristics from those of TCRalpha7.2- conventional T cells. They play important roles in various inflammatory diseases,including rheumatoid arthritis and inflammatory bowel disease. MAIT cells express a single T cell receptor alpha chain,TCRalpha7.2 segment associated with Jalpha33 and CDR3 with fixed length,which recognizes bacteria-derived vitamin B metabolites. However,the characteristics of MAIT cells and TCRalpha7.2+ CD161- T cells have never been compared. Here,we performed RNA sequencing to compare the properties of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. Genome-wide transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells,and TCRalpha7.2+ CD161- T cells were compared and analyzed using causal network analysis. This is the first report comparing the transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. We also identified the predominant signaling pathways of MAIT cells,which differed from those of TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells,through a gene set enrichment test and upstream regulator analysis and identified the genes responsible for the characteristic MAIT cell phenotypes. Our study advances the complete understanding of MAIT biology. View Publication