The transcription factor B Cell CLL/Lymphoma 11B (BCL11B) is indispensable for T lineage development of lymphoid progenitors. Here we show that chimeric antigen receptor (CAR) expression early in ex vivo generated lymphoid progenitors suppressed BCL11B, leading to suppression of T cell-associated gene expression and acquisition of natural killer (NK) cell-like properties. Upon adoptive transfer into hematopoietic stem cell transplant recipients they differentiated into CAR-induced killer cells (CARiK) that mediated potent antigen-directed antileukemic activity even across MHC barriers. A CD28 and active immune-receptor-tyrosine-based-activation-motifs were critical for a functional CARiK phenotype. These results give important insights into differentiation of murine and human lymphoid progenitors driven by synthetic CAR transgene-expression and encourage further evaluation of ex vivo generated CARiK cells for targeted immunotherapy.
Marcel Maluski, Arnab Ghosh, Jessica Herbst, Vanessa Scholl, Rolf Baumann, Jochen Huehn, Robert Geffers, Johann Meyer, Holger Maul, Britta Eiz-Vesper, Andreas Krueger, Axel Schambach, Marcel R.M. van den Brink, Martin G. Sauer
HIV is a major driver of Tuberculosis (TB) reactivation. Depletion of CD4+ T cells is assumed to be the basis behind TB reactivation in individuals with latent tuberculosis Infection (LTBI) co-infected with human immunodeficiency virus (HIV). Non-human primates (NHPs) coinfected with a mutant simian immunodeficiency virus (SIVΔGY), that does not cause depletion of tissue CD4+ T cells during infection, failed to reactivate TB. To investigate the contribution of CD4+ T cell depletion relative to other mechanisms of SIV-induced reactivation of LTBI, we used CD4R1 antibody to deplete CD4+ T cells in animals with LTBI without lentiviral infection. We showed that the mere depletion of CD4+ T cells during LTBI was insufficient in generating reactivation of LTBI. Instead, direct cytopathic effects of SIV resulting in chronic immune activation, along with the altered effector T cell phenotypes and dysregulated T cell homeostasis, were likely mediators of reactivation of LTBI. These results revealed important implications for controlling TB in the HIV co-infected individuals.
Allison N. Bucşan, Ayan Chatterjee, Dhiraj K. Singh, Taylor W. Foreman, Tae-Hyung Lee, Breanna Threeton, Melanie G. Kirkpatrick, Mushtaq Ahmed, Nadia Golden, Xavier Alvarez, James A. Hoxie, Smriti Mehra, Jyothi Rengarajan, Shabaana A. Khader, Deepak Kaushal
During developmental angiogenesis blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether and how cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates both cell death via apoptosis and necroptosis. Here we show that expression of Casp-8 in endothelial cells (ECs) was required for proper postnatal retina angiogenesis. EC specific Casp-8 knockout pups (Casp-8ECko) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting and migration independent of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 mitogen-activated protein kinase (MAPK) downstream of receptor-interacting serine/threonine- protein kinase 3 (RIPK3) and destabilization of VE-cadherin at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR), resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-8ECko pups. Taken together, we describe that Casp-8 acts in a cell-death independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP.
Nathalie Tisch, Aida Freire-Valls, Rosario Yerbes, Isidora Paredes, Silvia La Porta, Xiaohong Wang, Rosa Martín-Pérez, Laura Castro, Wendy Wei-Lynn Wong, Leigh Coultas, Boris Strilic, Hermann-Josef Gröne, Thomas Hielscher, Carolin Mogler, Ralf Adams, Peter Heiduschka, Lena Claesson-Welsh, Massimiliano Mazzone, Abelardo López-Rivas, Thomas Schmidt, Hellmut G. Augustin, Carmen Ruiz de Almodovar
Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH) defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO-signaling. We identified two pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin-toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.
Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer
Overexpression of myo-inositol oxygenase (MIOX), a proximal tubular enzyme, exacerbates cellular redox injury in acute kidney injury (AKI). Ferroptosis, a newly coined term associated with lipid hydroperoxidation, plays a critical role in the pathogenesis of AKI. Whether or not MIOX exacerbates tubular damage by accelerating ferroptosis in Cisplatin-induced AKI remains elusive. Cisplatin-treated HK-2 cells exhibited notable cell death, which was reduced by ferroptosis inhibitors. Also, alterations in various ferroptosis metabolic sensors, including lipid hydroperoxidation, glutathione peroxidase 4 (GPX4) activity, NADPH and reduced glutathione (GSH) levels, and ferritinophagy, were observed. These perturbations were accentuated by MIOX overexpression, while ameliorated by MIOX knockdown. Likewise, Cisplatin-treated CD1 mice exhibited tubular damage and derangement of renal physiological parameters, which was alleviated by Ferrostatin-1 (Fer-1), a ferroptosis inhibitor. To investigate the relevance of MIOX to ferroptosis, Wild-type (WT) mice, MIOX-overexpressing transgenic (MIOX-TG) mice and MIOX knockout (MIOX-KO) mice were subjected to Cisplatin treatment. In comparison to Cisplatin-treated WT mice, Cisplatin-treated MIOX-TG mice had more severe renal pathological changes and perturbations in ferroptosis metabolic sensors, which were minimal in Cisplatin-treated MIOX-KO mice. In conclusion, these findings indicate that ferroptosis, an integral process in the pathogenesis of Cisplatin-induced AKI, is modulated by the expression profile of MIOX.
Fei Deng, Isha Sharma, Yingbo Dai, Ming Yang, Yashpal S. Kanwar
The interleukin-3 receptor alpha subunit, CD123, is expressed on many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp’s ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.
Katsuhiro Togami, Timothy Pastika, Jason Stephansky, Mahmoud Ghandi, Amanda L. Christie, Kristen L. Jones, Carl A. Johnson, Ross W. Lindsay, Christopher L. Brooks, Anthony Letai, Jeffrey W. Craig, Olga Pozdnyakova, David M. Weinstock, Joan Montero, Jon C. Aster, Cory M. Johannessen, Andrew A. Lane
Asthma is a heterogeneous syndrome that has been subdivided into physiological phenotypes and molecular endotypes. The most specific phenotypic manifestation of asthma is indirect airway hyperresponsiveness (AHR), and a prominent molecular endotype is the presence of type-2 inflammation. The underlying basis for type-2 inflammation and its relationship to AHR are incompletely understood. We assessed the expression of type-2 cytokines in the airways of subjects with and without asthma who were extensively characterized for AHR. Using quantitative morphometry of the airway wall, we identified a shift in mast cells from the submucosa to the airway epithelium specifically associated with both type-2 inflammation and indirect AHR. Using ex vivo modeling of primary airway epithelial cells in organotypic co-culture with mast cells, we have shown that epithelial-derived IL-33 uniquely induced type-2 cytokines in mast cells, which regulated the expression of epithelial IL33 in a feedforward loop. This feedforward loop was accentuated in epithelial cells derived from subjects with asthma. These results demonstrate that type-2 inflammation and indirect AHR in asthma are related to a shift in mast cell infiltration to the airway epithelium, and that mast cells cooperate with epithelial cells through IL-33 signaling to regulate type-2 inflammation.
Matthew C. Altman, Ying Lai, James D. Nolin, Sydney Long, Chien-Chang Chen, Adrian M. Piliponsky, William A. Altemeier, Megan Larmore, Charles W. Frevert, Michael S. Mulligan, Steven F. Ziegler, Jason S. Debley, Michael C. Peters, Teal S. Hallstrand
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent auto-inflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients’ leukocytes and of PIGT-knockout THP-1 cells we show that, through increased IL-1β secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of auto-inflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also auto-inflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.
Britta Höchsmann, Yoshiko Murakami, Makiko Osato, Alexej Knaus, Michi Kawamoto, Norimitsu Inoue, Tetsuya Hirata, Shogo Murata, Markus Anliker, Thomas Eggermann, Marten Jäger, Ricarda Floettmann, Alexander Höellein, Sho Murase, Yasutaka Ueda, Jun-ichi Nishimura, Yuzuru Kanakura, Nobuo Kohara, Hubert Schrezenmeier, Peter M. Krawitz, Taroh Kinoshita
The parathyroid hormone receptor (PTH1R) mediates the biologic actions of parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP). Here, we showed that salt inducible kinases (SIKs) are key kinases that control the skeletal actions downstream of PTH1R and that this GPCR, when activated, inhibited cellular SIK activity. Sik gene deletion led to phenotypic changes that were remarkably similar to models of increased PTH1R signaling. In growth plate chondrocytes, PTHrP inhibited SIK3 and ablation of this kinase in proliferating chondrocytes rescued perinatal lethality of PTHrP-null mice. Combined deletion of Sik2/Sik3 in osteoblasts and osteocytes led to a dramatic increase in bone mass that closely resembled the skeletal and molecular phenotypes observed when these bone cells express a constitutively active PTH1R that causes Jansen’s metaphyseal chondrodysplasia. Finally, genetic evidence demonstrated that class IIa HDACs were key PTH1R-regulated SIK substrates in both chondrocytes and osteocytes. Taken together, our findings established that SIK inhibition is central to PTH1R action in bone development and remodeling. Furthermore, this work highlighted the key role of cAMP-regulated salt inducible kinases downstream of GPCR action.
Shigeki Nishimori, Maureen J. O'Meara, Christian Castro, Hiroshi Noda, Murat Cetinbas, Janaina da Silva Martins, Ugur Ayturk, Daniel J. Brooks, Michael Bruce, Mizuki Nagata, Wanida Ono, Christopher J. Janton, Mary L. Bouxsein, Marc Foretz, Rebecca Berdeaux, Ruslan I. Sadreyev, Thomas J. Gardella, Harald Jüppner, Henry M. Kronenberg, Marc N. Wein
The Toll-Like Receptor 8 (TLR8) has an important role in innate immune responses to RNA viral infections including respiratory syncytial virus (RSV). We reported previously that TLR8 expression was increased directly by the tumor suppressor and transcription factor p53 via a single nucleotide polymorphism (SNP: rs3761624) in the TLR8 promoter, thereby placing TLR8 in the p53/immune axis. Because this SNP is in linkage disequilibrium with other SNPs associated with several infectious diseases, we addressed the combined influence of p53 and the SNP on downstream inflammatory signaling in response to a TLR8 cognate ssRNA ligand. Using human primary lymphocytes, p53 induction by chemotherapeutic agents such as ionizing radiation caused SNP-dependent synergistic increases in IL-6 following incubation with an ssRNA ligand, as well as TLR8 RNA and protein expression along with p53 binding at the TLR-p53 SNP site. Because TLR8 is X-linked, the increases were generally reduced in heterozygous females. We found a corresponding association of the p53-responsive allele with RSV disease severity in infants hospitalized with RSV infection. We conclude that p53 can strongly influence TLR8 mediated immune responses and that knowledge of the p53 responsive SNP can inform diagnosis and prognosis of RSV disease and other diseases that might have a TLR8 component, including cancer.
Daniel Menendez, Joyce Snipe, Jacqui Marzec, Cynthia L. Innes, Fernando P. Polack, Mauricio Caballero, Shepherd H. Schurman, Steven R. Kleeberger, Michael A. Resnick
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