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1.
J Exp Med ; 220(12)2023 12 04.
Article in English | MEDLINE | ID: mdl-37796477

ABSTRACT

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.


Subject(s)
Programmed Cell Death 1 Receptor , Signal Transduction , Programmed Cell Death 1 Receptor/metabolism , Ligands , T-Lymphocytes/metabolism , Receptors, Antigen, T-Cell/metabolism , B7-H1 Antigen/metabolism
2.
Proc Natl Acad Sci U S A ; 119(33): e2208522119, 2022 08 16.
Article in English | MEDLINE | ID: mdl-35939714

ABSTRACT

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.


Subject(s)
Apoptosis , CD8-Positive T-Lymphocytes , Cytotoxicity, Immunologic , Fas Ligand Protein , T-Lymphocytes, Cytotoxic , Transcription Factors , Animals , Apoptosis/physiology , Chromatin/metabolism , Cytotoxicity, Immunologic/genetics , Fas Ligand Protein/metabolism , Granzymes/genetics , Humans , Mice , Perforin/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Science ; 376(6589): eabl5282, 2022 04 08.
Article in English | MEDLINE | ID: mdl-35389803

ABSTRACT

Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3-specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.


Subject(s)
Immunotherapy, Adoptive , Receptors, Antigen, T-Cell , T-Lymphocytes , Antigens, Neoplasm , Cross Reactions , Major Histocompatibility Complex , Myocardium/immunology , Peptides , T-Lymphocytes/metabolism
4.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Article in English | MEDLINE | ID: mdl-34588308

ABSTRACT

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.


Subject(s)
DNA/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Antigens/immunology , Cell Line, Tumor , Humans , Immunotherapy/methods , Jurkat Cells , Kinetics , Ligands , Lymphocyte Activation/immunology
5.
Immunohorizons ; 5(2): 117-132, 2021 02 23.
Article in English | MEDLINE | ID: mdl-33622708

ABSTRACT

Clustered regularly interspaced short palindromic repeats (CRISPR)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods. To overcome these challenges and limitations, we modified existing tools and developed three alternative methods for CRISPR-based genome editing using a hit-and-run transient expression strategy, together with a convenient system for promoting gene expression using CRISPRa. Overall, our CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering provide efficient alternatives to existing methods and have wide application not only in terms of hematopoietic cells but also other mammalian cell types of interest.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Gene Targeting/methods , Immune System , Animals , Humans , Lymphocytes/metabolism , Macrophages/metabolism , Mice
6.
Elife ; 82019 08 06.
Article in English | MEDLINE | ID: mdl-31385572

ABSTRACT

Despite existing evidence for tuning of innate immunity to different classes of bacteria, the molecular mechanisms used by macrophages to tailor inflammatory responses to specific pathogens remain incompletely defined. By stimulating mouse macrophages with a titration matrix of TLR ligand pairs, we identified distinct stimulus requirements for activating and inhibitory events that evoked diverse cytokine production dynamics. These regulatory events were linked to patterns of inflammatory responses that distinguished between Gram-positive and Gram-negative bacteria, both in vitro and after in vivo lung infection. Stimulation beyond a TLR4 threshold and Gram-negative bacteria-induced responses were characterized by a rapid type I IFN-dependent decline in inflammatory cytokine production, independent of IL-10, whereas inflammatory responses to Gram-positive species were more sustained due to the absence of this IFN-dependent regulation. Thus, disparate triggering of a cytokine negative feedback loop promotes tuning of macrophage responses in a bacteria class-specific manner and provides context-dependent regulation of inflammation dynamics.


Subject(s)
Feedback, Physiological , Gram-Negative Bacteria/immunology , Gram-Positive Bacteria/immunology , Interferon Type I/metabolism , Macrophages/immunology , Pneumonia, Bacterial/immunology , Animals , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Mice, Inbred C57BL
7.
J Biol Chem ; 291(50): 25921-25936, 2016 Dec 09.
Article in English | MEDLINE | ID: mdl-27777308

ABSTRACT

The activation of NF-κB downstream of T cell receptor (TCR) engagement is a key signaling step required for normal lymphocyte function during the adaptive immune response. During TCR signaling, the adaptor protein Bcl10 is inducibly recruited to the CARD11 scaffold protein as part of a multicomponent complex that induces IκB kinase (IKK) activity and NF-κB activation. Here, we show that a consequence of this recruitment is the TCR-induced conjugation of Bcl10 with linear-linked polyubiquitin chains to generate the signaling intermediate Lin(Ub)n-Bcl10, which is required for the association of Bcl10 with the NEMO subunit of the IKK complex. The TCR-induced generation of Lin(Ub)n-Bcl10 requires Bcl10 lysines 17, 31, and 63, CARD11, MALT1, and the HOIP subunit of the linear ubiquitin chain assembly complex (LUBAC) but not the HOIP accessory protein SHARPIN. CARD11 promotes signal-induced Lin(Ub)n-Bcl10 generation by co-recruiting Bcl10 with HOIP, thereby bringing substrate to enzyme. The CARD11-HOIP interaction is rendered TCR-inducible by the four autoinhibitory repressive elements in the CARD11 inhibitory domain and involves the CARD11 coiled-coil domain and two independent regions of HOIP. Interestingly, oncogenic CARD11 variants associated with diffuse large B cell lymphoma spontaneously induce Lin(Ub)n-Bcl10 production to extents that correlate with their abilities to activate NF-κB and with their enhanced abilities to bind HOIP and Bcl10. Our results define molecular determinants that control the production of Lin(Ub)n-Bcl10, an important signaling intermediate in TCR and oncogenic CARD11 signaling.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CARD Signaling Adaptor Proteins/metabolism , Guanylate Cyclase/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/physiology , Ubiquitination/physiology , Adaptor Proteins, Signal Transducing/genetics , B-Cell CLL-Lymphoma 10 Protein , CARD Signaling Adaptor Proteins/genetics , Caspases/genetics , Caspases/metabolism , Guanylate Cyclase/genetics , HEK293 Cells , Humans , Jurkat Cells , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Protein Domains , Receptors, Antigen, T-Cell/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
9.
Mol Cell Biol ; 36(5): 794-808, 2015 Dec 28.
Article in English | MEDLINE | ID: mdl-26711259

ABSTRACT

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.


Subject(s)
CARD Signaling Adaptor Proteins/metabolism , Guanylate Cyclase/metabolism , Lymphoma/metabolism , Receptors, Antigen/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , B-Cell CLL-Lymphoma 10 Protein , CARD Signaling Adaptor Proteins/chemistry , Cell Survival , Guanylate Cyclase/chemistry , HEK293 Cells , Humans , Jurkat Cells , NF-kappa B/metabolism , Protein Interaction Maps , Protein Structure, Tertiary , Ubiquitination
10.
J Neurosci Res ; 91(12): 1563-71, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24105649

ABSTRACT

Studies have demonstrated that embryonic cell therapy is a potential approach for the treatment of Huntington's disease (HD). However, because of the limited resource of embryos, greater attention is needed in developing more efficient surgical techniques that not only enhance the therapy outcome but also avoid inefficient therapeutics of transplantation. In this study, we explored the curative effects of two different transplantation methods using a rat model of HD. Whole ganglionic eminence (WGE) cells or phosphate-buffered saline were transplanted into unilateral striatum of quinolinic acid (QA)-lesioned rats using microtransplantation instruments (with an outer diameter of 50 µm) or traditional transplantation instruments (with an outer diameter of 470 µm). Apomorphine-induced rotation test and adjusting step test were assessed after QA-induced lesion and 2, 4, 6, 8, 10, and 12 weeks after transplantation. The expression of neuronal nuclei (NeuN), dopamine, cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32), and glial fibrillary acidic protein (GFAP) was analyzed at 12 weeks after transplantation. We observed that microtransplanted rats performed better in the stepping test and had higher numbers of DARPP-32-positive cells compared with traditionally transplanted rats. Moreover, microtransplantation group showed lower GFAP expression surrounding the grafts in unilateral striatum and a higher survival rate posttransplantation compared with the traditional transplantation group. We conclude that microtransplantation is capable of enhancing therapeutic efficacy in the rat model of HD. This finding establishes the basis of an alternative transplantation strategy for treatment of HD.


Subject(s)
Brain Tissue Transplantation/methods , Embryonic Stem Cells/transplantation , Huntington Disease/surgery , Stem Cell Transplantation/methods , Animals , Disease Models, Animal , Female , Rats , Rats, Sprague-Dawley
11.
Mol Cell Biol ; 33(2): 429-43, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23149938

ABSTRACT

Antigen receptor signaling to NF-κB, essential for normal lymphocyte activation, is dysregulated in several types of lymphoma. During normal signaling, the multidomain adapter CARD11 transitions from a closed, inactive state to an open, active scaffold that assembles a multiprotein complex, leading to NF-κB activation. The regulation of CARD11 scaffold function is bypassed by lymphoma-associated oncogenic CARD11 mutations that induce spontaneous signaling. We report an unbiased high-throughput quantitative signaling screen that identifies new CARD11 hyperactive variants and defines a LATCH domain that functions with the CARD to promote CARD11 autoinhibition. Gain-of-function mutations in the LATCH or CARD disrupt inhibitory domain binding, promote Bcl10 association, and induce Bcl10 ubiquitination, NF-κB activation, and human lymphoma cell survival. Our results identify CARD11 mutations with oncogenic potential, provide a mechanistic explanation for their signaling potency, and offer a straightforward method for the discovery of variants that promote the tumorigenesis of NF-κB-dependent lymphomas.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CARD Signaling Adaptor Proteins/genetics , Guanylate Cyclase/genetics , Lymphoma/genetics , Signal Transduction , Ubiquitination , Adaptor Proteins, Signal Transducing/genetics , Animals , B-Cell CLL-Lymphoma 10 Protein , CARD Signaling Adaptor Proteins/metabolism , Caspases/genetics , Caspases/metabolism , Cell Survival , Evaluation Studies as Topic , Gene Expression Regulation , Genetic Vectors , Guanylate Cyclase/metabolism , HEK293 Cells , High-Throughput Screening Assays , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Immunoprecipitation , Lymphocyte Activation/genetics , Lymphoma/metabolism , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Mice , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/metabolism , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Receptors, Antigen/genetics , Receptors, Antigen/metabolism
12.
J Immunol ; 183(10): 6095-101, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19841171

ABSTRACT

Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function. In this report we demonstrate that anergic T cells are metabolically anergic, in that upon full stimulation (signals 1 plus 2) they fail to up-regulate the machinery necessary to support increased metabolism. These findings suggest that one mechanism responsible for the maintenance of T cell anergy is failure to up-regulate the metabolic machinery. Furthermore, we demonstrate that by blocking leucine, glucose, and energy metabolism, T cell activation is mitigated. Additionally, inhibition of these metabolic pathways during T cell activation leads to anergy in Th1-differentiated cells. Overall, our findings extend the role of T cell metabolism in regulating T cell function.


Subject(s)
AMP-Activated Protein Kinases/immunology , Clonal Anergy , Energy Metabolism/drug effects , Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , AMP-Activated Protein Kinases/drug effects , AMP-Activated Protein Kinases/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , CD28 Antigens/immunology , CD3 Complex/immunology , Cell Line , Cytochromes c/immunology , Energy Metabolism/immunology , Glucose/immunology , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Immunologic Factors/pharmacology , Leucine/analogs & derivatives , Leucine/immunology , Leucine/metabolism , Lymphocyte Activation/drug effects , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/immunology , Mice , Receptors, Antigen, T-Cell/metabolism , Ribonucleotides/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Up-Regulation/drug effects , Up-Regulation/immunology
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