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1.
Nature ; 597(7877): 544-548, 2021 09.
Article in English | MEDLINE | ID: mdl-34526724

ABSTRACT

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.


Subject(s)
CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/drug effects , Cell Differentiation/drug effects , Drug Partial Agonism , Interleukin-2/analogs & derivatives , Interleukin-2/agonists , Mutant Proteins/pharmacology , Stem Cells/drug effects , Animals , CD8-Positive T-Lymphocytes/immunology , Interleukin-2/chemistry , Interleukin-2/genetics , Melanoma/metabolism , Mice , Mitochondria/drug effects , Mutant Proteins/chemistry , Mutant Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , STAT5 Transcription Factor/metabolism , Stem Cells/cytology , T Cell Transcription Factor 1/metabolism , Translational Research, Biomedical
2.
Trends Immunol ; 40(12): 1078-1081, 2019 12.
Article in English | MEDLINE | ID: mdl-31732285

ABSTRACT

The promise of natural killer (NK) cells as effectors in cancer cellular therapy is limited by their expression of dominant inhibitory receptors for human leukocyte antigen (HLA) class I. Here, we discuss how chimeric antigen receptors (CARs) engineered to override inhibitory signaling might boost NK cell antitumor responses, independently of blockade of NK cell inhibitory receptors.


Subject(s)
Cancer Vaccines/immunology , Immunotherapy, Adoptive/methods , Killer Cells, Natural/immunology , Neoplasms/therapy , Receptors, Chimeric Antigen/metabolism , Animals , Cytotoxicity, Immunologic , Genetic Engineering , HLA Antigens/metabolism , Humans , Killer Cells, Natural/transplantation , Neoplasms/immunology , Receptors, Chimeric Antigen/genetics , Signal Transduction , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation
3.
J Environ Sci (China) ; 94: 40-51, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32563486

ABSTRACT

Photo-responsive adsorption-photocatalysis nanocomposites are generally used in water and wastewater decontamination; however, the prolonged adsorption capacity of composites and the role of adsorption in concomitant photocatalysis are typically neglected. These composites can be regenerated under light irradiation as their adsorption capacity decreases. Herein, a novel adsorption-photocatalysis bifunctional nanocomposite, Bi-doped TiO2 supported on powdered activated carbon (Bi2O3/TiO2/PAC), was prepared using the sol-impregnation-hydrothermal procedure. Bi2O3/TiO2/PAC with a secondary calcination temperature of 700°C under a nitrogen atmosphere was selected for maximum adsorption capacity on Methyl Orange (MO). The composite displayed an excellent adsorption capacity and was easily separated and recycled. The results demonstrate that 71.2% photocatalytic regeneration efficiency could be attained under visible light irradiation for 1 hr at an intensity of 750 W/m2 and pH 7. Characterization of the as-prepared Bi2O3/TiO2/PAC nanocomposite (700°C) indicates that it possesses a highly specific surface area and great optical properties, showing bifunctional adsorption-photocatalysis characteristics. The p-n heterojunction of the composite played a dominant role in the photocatalytic regeneration process, and effective degradation of MO could be achieved along with composite regeneration.


Subject(s)
Azo Compounds , Titanium , Adsorption , Catalysis
4.
Nat Chem Biol ; 12(4): 226-32, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26829474

ABSTRACT

Faithful segregation of chromosomes in mammalian cells requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation, which triggers mitotic entry, have been extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via the acetyltransferase TIP60 in human cell division. CDK1-cyclin B phosphorylates Ser90 of TIP60, which elicits TIP60-dependent acetylation of Aurora B and promotes accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protects Aurora B's activation loop from dephosphorylation by the phosphatase PP2A to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling cascade that integrates protein phosphorylation and acetylation with cell cycle progression for maintenance of genomic stability.


Subject(s)
Aurora Kinase B/metabolism , Chromosome Segregation/physiology , Histone Acetyltransferases/metabolism , Kinetochores/enzymology , Mitosis/physiology , Acetylation , Antibodies, Monoclonal/pharmacology , Aurora Kinase B/genetics , Chromosome Segregation/genetics , Enzyme Inhibitors/pharmacology , HEK293 Cells , HeLa Cells , Histone Acetyltransferases/genetics , Humans , Immunoprecipitation , Kinetochores/ultrastructure , Lysine Acetyltransferase 5 , Mitosis/genetics , Plasmids , Time-Lapse Imaging
5.
J Biol Chem ; 291(39): 20692-706, 2016 09 23.
Article in English | MEDLINE | ID: mdl-27451391

ABSTRACT

Cell migration is orchestrated by dynamic interactions of microtubules with the plasma membrane cortex. How these interactions facilitate these dynamic processes is still being actively investigated. TIP150 is a newly characterized microtubule plus end tracking protein essential for mitosis and entosis (Ward, T., Wang, M., Liu, X., Wang, Z., Xia, P., Chu, Y., Wang, X., Liu, L., Jiang, K., Yu, H., Yan, M., Wang, J., Hill, D. L., Huang, Y., Zhu, T., and Yao, X. (2013) Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis. J. Biol. Chem. 288, 15771-15785; Xia, P., Zhou, J., Song, X., Wu, B., Liu, X., Li, D., Zhang, S., Wang, Z., Yu, H., Ward, T., Zhang, J., Li, Y., Wang, X., Chen, Y., Guo, Z., and Yao, X. (2014) Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction. J. Mol. Cell Biol. 6, 240-254). Here we show that TIP150 links dynamic microtubules to steer cell migration by interacting with cortactin. Mechanistically, TIP150 binds to cortactin via its C-terminal tail. Interestingly, the C-terminal TIP150 proline-rich region (CT150) binds to the Src homology 3 domain of cortactin specifically, and such an interaction is negatively regulated by EGF-elicited tyrosine phosphorylation of cortactin. Importantly, suppression of TIP150 or overexpression of phospho-mimicking cortactin inhibits polarized cell migration. In addition, CT150 disrupts the biochemical interaction between TIP150 and cortactin in vitro, and perturbation of the TIP150-cortactin interaction in vivo using a membrane-permeable TAT-CT150 peptide results in an inhibition of directional cell migration. We reason that a dynamic TIP150-cortactin interaction orchestrates directional cell migration via coupling dynamic microtubule plus ends to the cortical cytoskeleton.


Subject(s)
Cell Movement/physiology , Cortactin/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Cortactin/genetics , HEK293 Cells , Humans , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Protein Binding , src Homology Domains
6.
J Biol Chem ; 290(39): 23766-80, 2015 Sep 25.
Article in English | MEDLINE | ID: mdl-26242911

ABSTRACT

Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Movement/physiology , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , ras GTPase-Activating Proteins/metabolism , Amino Acid Motifs , Cell Cycle Proteins/genetics , Cell Movement/drug effects , Epidermal Growth Factor/pharmacology , HEK293 Cells , Humans , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Protein Binding , Protein Structure, Tertiary , ras GTPase-Activating Proteins/genetics
7.
Cell Rep ; 43(4): 114105, 2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38619967

ABSTRACT

Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.


Subject(s)
Killer Cells, Natural , Polysaccharides , Humans , Polysaccharides/metabolism , Killer Cells, Natural/metabolism , Killer Cells, Natural/immunology , Amino Sugars/metabolism , Genomics/methods , Rituximab/pharmacology , Rituximab/metabolism , Cell Line, Tumor
8.
Cancer Cell ; 42(2): 238-252.e9, 2024 02 12.
Article in English | MEDLINE | ID: mdl-38215749

ABSTRACT

Diffuse large B cell lymphoma (DLBCL) is an aggressive, profoundly heterogeneous cancer, presenting a challenge for precision medicine. Bruton's tyrosine kinase (BTK) inhibitors block B cell receptor (BCR) signaling and are particularly effective in certain molecular subtypes of DLBCL that rely on chronic active BCR signaling to promote oncogenic NF-κB. The MCD genetic subtype, which often acquires mutations in the BCR subunit, CD79B, and in the innate immune adapter, MYD88L265P, typically resists chemotherapy but responds exceptionally to BTK inhibitors. However, the underlying mechanisms of response to BTK inhibitors are poorly understood. Herein, we find a non-canonical form of chronic selective autophagy in MCD DLBCL that targets ubiquitinated MYD88L265P for degradation in a TBK1-dependent manner. MCD tumors acquire genetic and epigenetic alterations that attenuate this autophagic tumor suppressive pathway. In contrast, BTK inhibitors promote autophagic degradation of MYD88L265P, thus explaining their exceptional clinical benefit in MCD DLBCL.


Subject(s)
Lymphoma, Large B-Cell, Diffuse , Humans , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Myeloid Differentiation Factor 88/pharmacology , Signal Transduction , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Autophagy
9.
J Biol Chem ; 287(47): 39380-90, 2012 Nov 16.
Article in English | MEDLINE | ID: mdl-23035123

ABSTRACT

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent study shows that mitotic motor CENP-E cooperates with SKAP to orchestrate an accurate chromosome movement in mitosis. However, it remains elusive how kinetochore core microtubule binding activity KMN (KNL1-MIS12-NDC80) regulates microtubule plus-end dynamics. Here, we identify a novel interaction between MIS13 and SKAP that orchestrates accurate interaction between kinetochore and dynamic spindle microtubules. SKAP physically interacts with MIS13 and specifies kinetochore localization of SKAP. Suppression of MIS13 by small interfering RNA abrogates the kinetochore localization of SKAP. Total internal reflection fluorescence microscopic assays demonstrate that SKAP exhibits an EB1-dependent, microtubule plus-end loading and tracking in vitro. Importantly, SKAP is essential for kinetochore oscillations and dynamics of microtubule plus-ends during live cell mitosis. Based on those findings, we reason that SKAP constitutes a dynamic link between spindle microtubule plus-ends and mitotic chromosomes to achieve faithful cell division.


Subject(s)
Cell Cycle Proteins/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Mitosis/physiology , Multiprotein Complexes/metabolism , Spindle Apparatus/metabolism , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , HeLa Cells , Humans , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Multiprotein Complexes/genetics , Spindle Apparatus/genetics
10.
J Biol Chem ; 287(2): 1500-9, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22110139

ABSTRACT

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation/physiology , Chromosomes, Human/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Mitosis/physiology , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Human/genetics , HeLa Cells , Humans , Kinesins/genetics , Kinesins/metabolism , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Microtubules/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
11.
J Inflamm Res ; 16: 2607-2613, 2023.
Article in English | MEDLINE | ID: mdl-37360623

ABSTRACT

Objective: To investigate the changes in the proportion of peripheral blood lymphocytes and the expression of HLA II molecules in lymphocytes during acute rejection after renal transplantation. Methods: Thirty-five patients who had undergone renal transplantation were selected. Eighteen patients with clinical and pathological confirmed acute rejection were selected as the test group, and twelve patients without clinical acute rejection symptoms were selected as the control group. Flow cytometry analysis was used to determine the proportion of peripheral blood lymphocytes. The mRNA and protein expression of HLA II molecules on peripheral blood lymphocytes were detected using real-time fluorescence quantification and immunoblotting, respectively. Results: The proportion of T lymphocytes, B lymphocytes, and CD4CD8 double positive T cells in the Control Group were 67.48% ± 5.35%, 10.82% ± 1.26%, and 0.88% ± 0.06%, respectively, and in the Test Group were 87.52% ± 6.28%, 3.36% ± 0.26%, and 0.34% ± 0.03%, with a significant difference respectively. The mRNA and protein expressions of HLA II molecules of peripheral blood B lymphocytes in the control group were significantly higher that these in the test group. Conclusion: The proportion of peripheral blood T lymphocytes, B lymphocytes, CD4CD8 double positive T cells, and the expression of HLA II molecules of peripheral blood lymphocytes can all indicate the occurrence of acute renal transplantation rejection, which were exceedingly useful to clinicians in judging the acute rejection of renal transplantation in the early stages.

12.
Front Immunol ; 13: 840844, 2022.
Article in English | MEDLINE | ID: mdl-35585985

ABSTRACT

Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness, a process termed licensing or education, but hinder their ability to kill HLA-I+ tumor cells. Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I+ tumor cells. As proof of concept, we show here that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I+ tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog, TMIGD2) and 2B4 (CD244, SLAMF4). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody, the CD28H transmembrane domain, and the fusion of CD28H, 2B4, and TCRζ signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCRζ signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore, such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore, engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies, such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Adoptive Transfer , CD28 Antigens , Cytotoxicity, Immunologic , Humans , Immunotherapy, Adoptive , Killer Cells, Natural , Neoplasms/therapy , Receptors, Chimeric Antigen/genetics
13.
Methods Mol Biol ; 2463: 269-288, 2022.
Article in English | MEDLINE | ID: mdl-35344181

ABSTRACT

Natural killer (NK) cells are an important component of the cancer immune surveillance system. They are regulated by germline-encoded receptors that activate and inhibit their effector function, such as secretion of cytokines and direct lysis of tumor cells and virus-infected cells. Without the need to be primed by prior exposure to tumor antigen, NK cells can detect ligands expressed on tumor cells and selectively kill these cells. NK cells are under strict control by inhibitory receptors that bind to HLA class I on target cells and block early activation signals, thus preventing lysis of target cells. The sensitivity to lysis by NK cells is therefore determined to a large extent by the expression of HLA class I molecules on tumor cells. In addition to receptor-ligand interactions that occur at NK-target cell synapses, many other factors determine the sensitivity of tumor cells to lysis by NK. Intrinsic properties of tumor cells, such as their metabolism and signaling networks establish a threshold above which they will succumb to the death pathways triggered by NK cell attack. Here we provide a protocol for a genome-wide CRISPR screen in tumor cells to identify factors that regulate their sensitivity to primary human NK cells. Tumor cells first transduced for expression of Cas9 are then transduced with a guide RNA (gRNA) library and co-cultured with NK cells. Deep sequencing of the library generated from the genome of tumor cells that survived the selection by NK cells and analysis of the distribution of guide RNAs is performed to identify genes that promote either sensitivity or resistance to NK-mediated killing. The contribution of individual genes to tumor sensitivity can be validated by knockouts using individual gRNAs. The techniques and workflow described here could be applied to primary tumors from cancer patients and reveal tumor-specific points of vulnerability that could be exploited for cancer immunotherapy, such as checkpoint blockade or expression of chimeric antigen receptors specifically designed to activate NK cell cytotoxicity.


Subject(s)
Killer Cells, Natural , Neoplasms , Cell Count , Humans , Immunotherapy , Neoplasms/genetics , Signal Transduction
14.
J Mol Cell Biol ; 13(12): 841-852, 2022 01 29.
Article in English | MEDLINE | ID: mdl-34554241

ABSTRACT

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP-Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore-microtubule attachments to achieve faithful cell division.


Subject(s)
Cell Cycle Proteins , Microtubule-Associated Proteins , Aurora Kinase B , Cell Cycle Proteins/genetics , Chromosome Segregation , HeLa Cells , Humans , Kinetochores , Microtubule-Associated Proteins/genetics , Microtubules , Mitosis
15.
Cancer Immunol Res ; 7(6): 939-951, 2019 06.
Article in English | MEDLINE | ID: mdl-31018957

ABSTRACT

The CD28-B7 family of receptor-ligand pairs regulates lymphocyte responses through costimulation and coinhibition. It includes checkpoint inhibitors, such as PD-1, which limit antitumor and antivirus T-cell responses. CD28 homolog (CD28H) and B7H7 have been identified as a receptor-ligand pair in this family, which has costimulatory activity in T cells. Here, we show that CD28H is expressed in primary natural killer (NK) cells and that it is a strong activator of NK cells through selective synergy with receptors NKp46 and 2B4 to induce degranulation, lysis of target cells, and production of proinflammatory cytokines. Expression of B7H7 on target cells enhanced both natural and antibody-dependent cellular cytotoxicity of NK cells. Mutation of tyrosine 192 on the CD28H cytoplasmic tail abolished NK-cell activation through CD28H. As B7H7 is broadly expressed in tumor tissues, we engineered a CD28H chimeric antigen receptor (CD28H-CAR) consisting of full-length CD28H fused to the cytoplasmic domain of T-cell receptor ζ chain. Remarkably, expression of CD28H-CAR in NK cells triggered lysis of B7H7+ HLA-E+ tumor cells by overriding inhibition by the HLA-E receptor NKG2A. The cytoplasmic domains of CD28H and of the ζ chain were both required for this activity. Thus, CD28H is a powerful activation receptor of NK cells that broadens their antitumor activity and holds promise as a component of NK-based CARs for cancer immunotherapy.


Subject(s)
B7 Antigens/immunology , CD28 Antigens/metabolism , Cytotoxicity, Immunologic , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocyte Activation/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Biomarkers , CD28 Antigens/genetics , Cell Degranulation/immunology , Cytokines/biosynthesis , Humans , Immunophenotyping , Immunotherapy, Adoptive , NK Cell Lectin-Like Receptor Subfamily C/metabolism , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism
16.
Front Immunol ; 10: 2879, 2019.
Article in English | MEDLINE | ID: mdl-31921143

ABSTRACT

The anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on IFNGR2. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.


Subject(s)
Carcinoma, Renal Cell , Interferon-gamma , Killer Cells, Natural/immunology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Tumor Escape , B7 Antigens/genetics , B7 Antigens/immunology , CRISPR-Cas Systems , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/immunology , Carcinoma, Renal Cell/pathology , Genome-Wide Association Study , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , K562 Cells , Kidney Neoplasms/genetics , Kidney Neoplasms/immunology , Kidney Neoplasms/pathology , Killer Cells, Natural/pathology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Natural Cytotoxicity Triggering Receptor 3/genetics , Natural Cytotoxicity Triggering Receptor 3/immunology , Receptors, Interferon/genetics , Receptors, Interferon/immunology
17.
J Mol Cell Biol ; 10(6): 527-538, 2018 12 01.
Article in English | MEDLINE | ID: mdl-30016453

ABSTRACT

Heterochromatin protein 1α (HP1α) regulates chromatin specification and plasticity during cell fate decision. Different structural determinants account for HP1α localization and function during cell division cycle. Our earlier study showed that centromeric localization of HP1α depends on the epigenetic mark H3K9me3 in interphase, while its centromeric location in mitosis relies on uncharacterized PXVXL-containing factors. Here, we identified a PXVXL-containing protein, ligand-dependent nuclear receptor-interacting factor 1 (LRIF1), which recruits HP1α to the centromere of mitotic chromosomes and its interaction with HP1α is essential for accurate chromosome segregation during mitosis. LRIF1 interacts directly with HP1α chromoshadow domain via an evolutionarily conserved PXVXL motif within its C-terminus. Importantly, the LRIF1-HP1α interaction is critical for Aurora B activity in the inner centromere. Mutation of PXVXL motif of LRIF1 leads to defects in HP1α centromere targeting and aberrant chromosome segregation. These findings reveal a previously unrecognized direct link between LRIF1 and HP1α in centromere plasticity control and illustrate the critical role of LRIF1-HP1α interaction in orchestrating accurate cell division.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Mitosis , Telomere-Binding Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Line , Centromere/metabolism , Centromere/ultrastructure , Chromobox Protein Homolog 5 , HeLa Cells , Humans , Protein Interaction Maps
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