B-cell-directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates.

ABSTRACT: Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

RATIONALE: Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. OBJECTIVES: To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. METHODS: In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. RESULTS: GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. CONCLUSIONS: GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease.

Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic-plus-binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain-containing mTOR-interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic-plus-binge ethanol-fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute-on-chronic liver injury. Mechanistically, the lipid-lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1 (SREBP-1). DEPTOR-dependent inhibition of mTORC1 also attenuated alcohol-induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol-mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up-regulate lipogenesis, to down-regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic-plus-binge ethanol feeding led to activation of SREBP-1 and lipin 1 through S6K1-dependent and independent mechanisms. Furthermore, hepatocyte-specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice. CONCLUSION: The dysregulation of SIRT1-DEPTOR-mTORC1 signaling is a critical determinant of ALD pathology; targeting SIRT1 and DEPTOR and selectively inhibiting mTORC1-S6K1 signaling may have therapeutic potential for treating ALD in humans. (Hepatology 2018).

The α-subunit regulates stability of the metal ion at the ligand-associated metal ion-binding site in ß3 integrins.

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin ßA domain of the ß-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two ß3 integrins αIIbß3 and αVß3, but a potential role of this interaction on stability of the metal ion at LIMBS in ß3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated ß3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbß3 versus αVß3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVß3. Replacing the αIIb-LIMBS interface residue Phe(191) in αIIb (equivalent to Trp(179) in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbß3; a Trp(179) to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbß3 and a W179/F substitution in αVß3 reduced and increased, respectively, the apparent affinity of Mn(2+) to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVß3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVß3 ectodomain-17E6 Fab complex.

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVß3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVß3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

The Asthma Risk Gene, GSDMB, Promotes Mitochondrial DNA-induced ISGs Expression.

Released mitochondrial DNA (mtDNA) in cells activates cGAS-STING pathway, which induces expression of interferon-stimulated genes (ISGs) and thereby promotes inflammation, as frequently seen in asthmatic airways. However, whether the genetic determinant, Gasdermin B (GSDMB), the most replicated asthma risk gene, regulates this pathway remains unknown. We set out to determine whether and how GSDMB regulates mtDNA-activated cGAS-STING pathway and subsequent ISGs induction in human airway epithelial cells. Using qPCR, ELISA, native polyacrylamide gel electrophoresis, co-immunoprecipitation and immunofluorescence assays, we evaluated the regulation of GSDMB on cGAS-STING pathway in both BEAS-2B cells and primary normal human bronchial epithelial cells (nHBEs). mtDNA was extracted in plasma samples from human asthmatics and the correlation between mtDNA levels and eosinophil counts was analyzed. GSDMB is significantly associated with RANTES expression in asthmatic nasal epithelial brushing samples from the Genes-environments and Admixture in Latino Americans (GALA) II study. Over-expression of GSDMB promotes DNA-induced IFN and ISGs expression in bronchial epithelial BEAS-2B cells and nHBEs. Conversely, knockout of GSDMB led to weakened induction of interferon (IFNs) and ISGs in BEAS-2B cells. Mechanistically, GSDMB interacts with the C-terminus of STING, promoting the translocation of STING to Golgi, leading to the phosphorylation of IRF3 and induction of IFNs and ISGs. mtDNA copy number in serum from asthmatics was significantly correlated with blood eosinophil counts especially in male subjects. GSDMB promotes the activation of mtDNA and poly (dA:dT)-induced activation of cGAS-STING pathway in airway epithelial cells, leading to enhanced induction of ISGs.

Notch signaling drives intestinal graft-versus-host disease in mice and nonhuman primates.

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Identification and Tracking of Alloreactive T Cell Clones in Rhesus Macaques Through the RM-scTCR-Seq Platform.

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell's transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, 'RM-scTCR-Seq', which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR's with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.

Contributions of specificity protein-1 and steroidogenic factor 1 to Adcy4 expression in Y1 mouse adrenal cells.

The type 4 adenylyl cyclase, Adcy4, is the least abundant of five different adenylyl cyclase isoforms expressed in the Y1 mouse adrenocortical cell line and is deficient in a Y1 mutant with impaired steroidogenic factor 1 (SF1) activity. This study examines the contributions of SF1 and other DNA promoter/regulatory elements to Adcy4 expression in the Y1 cell line and its derivative Adcy4-deficient mutant. Primer extension and in silico analyses indicate that Adcy4 transcription initiates from multiple sites just downstream of a GC-rich sequence. Luciferase reporter gene assays identify a 124-bp sequence, situated 19 bp upstream of the major transcription start site and highly conserved among several mammalian species, as the major determinant of Adcy4 expression in Y1 cells and as a site of compromised activity in the Adcy4-deficient mutant. EMSAs using competitor nucleotides and specific antibodies indicate that this conserved region contains three specificity protein (Sp)-1/Sp3-binding sites and one SF1-binding site. As determined by site-specific mutagenesis, the 5'-most Sp1/Sp3-site enhances promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress Adcy4 promoter activity. In the Adcy4-deficient mutant, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with small interfering RNAs increases Adcy4 expression, confirming the contribution of SF1 to the mutant phenotype. These studies demonstrate roles for Sp1/Sp3 and SF1 in Adcy4 expression in Y1 cells and establish a repressor function for SF1 in certain promoter contexts.

AMPK Activation by Metformin Suppresses Abnormal Extracellular Matrix Remodeling in Adipose Tissue and Ameliorates Insulin Resistance in Obesity.

Fibrosis is emerging as a hallmark of metabolically dysregulated white adipose tissue (WAT) in obesity. Although adipose tissue fibrosis impairs adipocyte plasticity, little is known about how aberrant extracellular matrix (ECM) remodeling of WAT is initiated during the development of obesity. Here we show that treatment with the antidiabetic drug metformin inhibits excessive ECM deposition in WAT of ob/ob mice and mice with diet-induced obesity, as evidenced by decreased collagen deposition surrounding adipocytes and expression of fibrotic genes including the collagen cross-linking regulator LOX Inhibition of interstitial fibrosis by metformin is likely attributable to the activation of AMPK and the suppression of transforming growth factor-ß1 (TGF-ß1)/Smad3 signaling, leading to enhanced systemic insulin sensitivity. The ability of metformin to repress TGF-ß1-induced fibrogenesis is abolished by the dominant negative AMPK in primary cells from the stromal vascular fraction. TGF-ß1-induced insulin resistance is suppressed by AMPK agonists and the constitutively active AMPK in 3T3L1 adipocytes. In omental fat depots of obese humans, interstitial fibrosis is also associated with AMPK inactivation, TGF-ß1/Smad3 induction, aberrant ECM production, myofibroblast activation, and adipocyte apoptosis. Collectively, integrated AMPK activation and TGF-ß1/Smad3 inhibition may provide a potential therapeutic approach to maintain ECM flexibility and combat chronically uncontrolled adipose tissue expansion in obesity.

Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4.

Four mutant clones independently derived from the Y1 mouse adrenocortical tumor cell line have adenylyl cyclase (AC) activities that are resistant to forskolin, a direct activator of AC. In this study the AC isoform composition of the forskolin-resistant mutants was examined in order to explore the underlying basis for the resistance to forskolin. As determined by Western blot and RT-PCR analysis, the four forskolin-resistant mutants all were deficient in AC-4; the levels of other AC isoforms (AC-1, AC-3 and AC-5/6) were comparable to the levels in parent Y1 cells. Transfection of one of the mutant clones with an AC-4 expression vector increased forskolin-stimulated cAMP signaling, and restored forskolin-induced changes in cell morphology and growth. Taken together, these observations indicate that AC-4 deficiency is a hallmark of the forskolin-resistant phenotype of these mutants and suggest that AC-4 is an important target of forskolin action in the Y1 adrenal cell line.

Expression of adenylyl cyclase-4 (AC-4) in Y1 and forskolin-resistant adrenal cells.

Forskolin-resistant mutants of a mouse adrenocortical cell line present a complex phenotype in which adenylyl cyclase (AC) is resistant to activation by forskolin and by ACTH. ACTH-resistance results from a defect affecting transcription of the ACTH receptor and can be overcome by transfecting mutant cells with expression vectors encoding G beta/gamma. Forskolin-resistance results from an AC-4 deficiency. We now demonstrate that the AC-4 deficiency in forskolin-resistant mutants results from a transcription defect affecting the promoter activity of the AC-4 gene. Furthermore, the underlying defect leading to AC-4 deficiency and forskolin-resistance can be overcome by transfection of mutant clones with expression vectors encoding G beta/gamma. These data support our hypothesis that AC-4 is a preferred target of forskolin action in Y1 cells, demonstrate novel roles for G beta/gamma in gene expression and indicate that a common underlying defect, suppressible by G beta/gamma, accounts for both the resistance to ACTH and to forskolin.

Simultneous Expression of CS3 Colonization Factor Antigen and Cholera Toxin B Subunit in Salmonella typhi.

A host-plasmid balancing system was established based on asd gene in an avirulent strain of Salmonella typhi to express enterotoxigenic E.coli surface antigen CS3 and V.cholerae toxin subunit B(CTB). The plasmid can be stably maintained in the host and can express CS3 and CTB in the host cell without any antibiotic selection, although expression level and growth characteristics of the recombinant strain expressing either CS3 or CTB are superior to that of the recombinant strain which expresses both of the antigens. Antibo-dies against CS3 and CTB can be detected in sera of mice immunized with recombinant bacteria either orally or subcutaneously, and mice immunized subcutaneously can be protected from challenging with virulent strain of Salmonella typhi. This work may be helpful in constructing multivalent recombinant vaccines for prevention of bacterial diarrhea.

Structural basis for pure antagonism of integrin αVß3 by a high-affinity form of fibronectin.

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVß3 bound to a physiologic ligand, the tenth type III RGD domain of wild-type fibronectin (wtFN10), or to a high-affinity mutant (hFN10) shown here to act as a pure antagonist. Comparison of these structures revealed a central π-π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the ß3 subunit that blocked conformational changes triggered by wtFN10 and trapped hFN10-bound αVß3 in an inactive conformation. Removing the Trp1496 or Tyr122 side chains or reorienting Trp1496 away from Tyr122 converted hFN10 into a partial agonist. These findings offer new insights into the mechanism of integrin activation and a basis for the design of RGD-based pure antagonists.

Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta transmembrane fragment.

We determined the crystal structure of 1TM-alphaVbeta3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the alphaV and beta3 subunits. 1TM-alphaVbeta3 is more compact and less active in solution when compared with DeltaTM-alphaVbeta3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the alpha-beta interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length alphaVbeta3 on live cells yields a donor-membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2-thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.