While the impact of T helper 17 (Th17) cells in autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We have discovered SNARE complex proteins in Th17 cells enabling a vesicular glutamate release pathway inducing local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of β1-integrin to VCAM-1 on neurons in inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, known to be linked to integrin expression and highly expressed on stimulated T cells. While KV1.3 is not expressed in the CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from multiple sclerosis patients secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that β1-integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.
Katharina Birkner, Beatrice Wasser, Tobias Ruck, Carine Thalman, Dirk Luchtman, Katrin Pape, Samantha Schmaul, Lynn Bitar, Eva-Maria Krämer-Albers, Albrecht Stroh, Sven G. Meuth, Frauke Zipp, Stefan Bittner
Efficacy of dendritic cell (DC) cancer vaccines is classically thought to depend on their antigen-presenting cell (APC) activity. Studies show, however, that DC vaccine priming of cytotoxic T lymphocytes (CTL) requires the activity of endogenous DC, suggesting that exogenous DC stimulate anti-tumor immunity by transferring antigen (Ag) to endogenous DC. Such Ag transfer functions are most commonly ascribed to monocytes, implying that undifferentiated monocytes would function equally well as a vaccine modality and need not be differentiated to DC to be effective. Here, we used several murine cancer models to test the anti-tumor efficacy of undifferentiated monocytes loaded with protein or peptide Ag. Intravenously injected monocytes displayed anti-tumor activity superior to DC vaccines in several cancer models, including aggressive intracranial glioblastoma. Ag-loaded monocytes induced robust CTL responses via Ag transfer to splenic CD8+ DC in a manner independent of monocyte APC activity. Ag transfer required cell-cell contact and the formation of connexin 43-containing gap junctions between monocytes and DC. These findings demonstrate the existence of an efficient gap junction-mediated Ag transfer pathway between monocytes and CD8+ DC and suggest that administration of tumor Ag-loaded undifferentiated monocytes may serve as a simple and efficacious immunotherapy for the treatment of human cancers.
Min-Nung Huang, Lowell T. Nicholson, Kristen A. Batich, Adam M. Swartz, David Kopin, Sebastian Wellford, Vijay K. Prabhakar, Karolina Woroniecka, Smita K. Nair, Peter E. Fecci, John H. Sampson, Michael D. Gunn
Iron deficiency is common worldwide and is associated with adverse pregnancy outcomes. The increasing prevalence of indiscriminate iron supplementation during pregnancy also raises concerns about the potential adverse effects of iron excess. We examined how maternal iron status affects the delivery of iron to the placenta and fetus. Using mouse models, we documented maternal homeostatic mechanisms which protect the placenta and fetus from maternal iron excess. We determined that under physiological conditions or in iron deficiency, fetal and placental hepcidin does not regulate fetal iron endowment. With maternal iron deficiency, critical transporters mediating placental iron uptake (transferrin receptor 1, TFR1) and export (ferroportin, FPN) were strongly regulated. In mice, not only was TFR1 increased but FPN was surprisingly decreased to preserve placental iron, in the face of fetal iron deficiency. In human placentas from pregnancies with mild iron deficiency, TFR1 was increased but without a change in FPN. However, induction of more severe iron deficiency in human trophoblast in vitro resulted in the regulation of both TFR1 and FPN, similarly to the mouse model. This placental adaptation prioritizing placental iron is mediated by the iron-regulatory protein 1 and is important for the maintenance of mitochondrial respiration, thus ultimately protecting the fetus from the potentially dire consequences of generalized placental dysfunction.
Veena Sangkhae, Allison L. Fisher, Shirley Wong, Mary Dawn Koenig, Lisa Tussing-Humphreys, Alison Chu, Melisa Lelić, Tomas Ganz, Elizabeta Nemeth
CD8+ T cell responses are necessary for immune control of simian immunodeficiency virus (SIV). However, the key parameters that dictate antiviral potency remain elusive, conceivably because most studies to date have been restricted to analyses of circulating CD8+ T cells. We conducted a detailed clonotypic, functional, and phenotypic survey of SIV-specific CD8+ T cells across multiple anatomical sites in chronically infected rhesus macaques with high (> 10,000 copies/mL plasma) or low burdens of viral RNA (< 10,000 copies/mL plasma). No significant differences in response magnitude were identified across anatomical compartments. Rhesus macaques with low viral loads (VLs) harbored higher frequencies of polyfunctional CXCR5+ SIV-specific CD8+ T cells in various lymphoid tissues and higher proportions of unique Gag-specific CD8+ T cell clonotypes in the mesenteric lymph nodes relative to rhesus macaques with high VLs. In addition, public Gag-specific CD8+ T cell clonotypes were more commonly shared across distinct anatomical sites than the corresponding private clonotypes, which tended to form tissue-specific repertoires, especially in the peripheral blood and the gastrointestinal tract. Collectively, these data suggest that functionality and tissue localization are important determinants of CD8+ T cell-mediated efficacy against SIV.
Carly E. Starke, Carol L. Vinton, Kristin Ladell, James E. McLaren, Alexandra M. Ortiz, Joseph C. Mudd, Jacob K. Flynn, Stephen H. Lai, Fan Wu, Vanessa M. Hirsch, Samuel Darko, Daniel C. Douek, David A. Price, Jason M. Brenchley
β-thalassemia is a genetic anemia caused by partial or complete loss of β-globin synthesis leading to ineffective erythropoiesis and RBCs with short life-span. Currently, there is no efficacious oral medication modifying anemia for patients with beta-thalassemia. The inappropriately low levels of the iron regulatory hormone hepcidin enable excessive iron absorption by ferroportin, the unique cellular iron exporter in mammals, leading to organ iron overload and associated morbidities. Correction of unbalanced iron absorption and recycling by induction of hepcidin synthesis or treatment with hepcidin mimetics ameliorates β-thalassemia. However, hepcidin modulation or replacement strategies currently in clinical development all require parenteral drug administration. We identified oral ferroportin inhibitors by screening a library of small molecular weight compounds for modulators of ferroportin internalization. Restricting iron availability by VIT-2763, the first clinical stage oral ferroportin inhibitor, ameliorated anemia and the dysregulated iron homeostasis in the Hbbth3/+ mouse model of beta-thalassemia intermedia. VIT-2763 not only improved erythropoiesis but also corrected the proportions of myeloid precursors in spleens of Hbbth3/+ mice. VIT-2763 is currently developed as an oral drug targeting ferroportin for the treatment of β-thalassemia.
Vania Manolova, Naja Nyffenegger, Anna Flace, Patrick Altermatt, Ahmet Varol, Cédric Doucerain, Hanna Sundstrom, Franz Dürrenberger
Vascular Ehlers-Danlos syndrome (vEDS) is an autosomal-dominant connective tissue disorder caused by heterozygous mutations in the COL3A1 gene, which encodes the pro-alpha 1 chain of collagen III. Loss of structural integrity of the extracellular matrix is believed to drive the signs and symptoms of this condition, including spontaneous arterial dissection and/or rupture, the major cause of mortality. We created two mouse models of vEDS that carry heterozygous mutations in Col3a1 that encode glycine substitutions analogous to those found in patients, and showed that signaling abnormalities in the PLC/IP3/PKC/ERK pathway (phospholipase C/inositol 1,4,5-triphosphate/protein kinase C/extracellular signal-regulated kinase) are major mediators of vascular pathology.Treatment with pharmacologic inhibitors of ERK1/2 or PKC-beta prevented death due to spontaneous aortic rupture. Additionally, we found that pregnancy- and puberty-associated accentuation of vascular risk, also seen in vEDS patients, is rescued by attenuation of oxytocin and androgen signaling, respectively. Taken together, our results provide evidence that targetable signaling abnormalities contribute to the pathogenesis of vEDS, highlighting unanticipated therapeutic opportunities.
Caitlin J. Bowen, Juan Francisco Calderón Giadrosic, Zachary Burger, Graham Rykiel, Elaine C. Davis, Mark R. Helmers, Kelly Benke, Elena Gallo MacFarlane, Harry C. Dietz
Recent occurrences of filoviruses and the arenavirus Lassa virus (LASV) in overlapping endemic areas of Africa highlight the need for a prophylactic vaccine that would confer protection against all of these viruses that cause lethal hemorrhagic fever (HF). We developed a quadrivalent formulation of Vesiculovax that contains recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins and also contains a rVSV vector expressing the glycoprotein of a lineage IV strain of LASV. Cynomolgus macaques were vaccinated twice with the quadrivalent formulation, followed by challenge 28 days after the boost vaccination with each of the three corresponding filoviruses (Ebola, Sudan, Marburg) or a heterologous contemporary lineage II strain of LASV. Serum IgG and neutralizing antibody responses specific for all four glycoproteins were detected in all vaccinated animals. A modest and balanced cell-mediated immune response specific for the glycoproteins was also detected in most of the vaccinated macaques. Regardless of the levels of total glycoprotein-specific immune response detected after vaccination, all immunized animals were protected from disease and death following lethal challenges. These findings indicate that vaccination with attenuated rVSV vectors each expressing a single HF virus glycoprotein may provide protection against those filoviruses and LASV most commonly responsible for outbreaks of severe HF in Africa.
Robert W. Cross, Rong Xu, Demetrius Matassov, Stefan Hamm, Theresa E. Latham, Cheryl S. Gerardi, Rebecca M. Nowak, Joan B. Geisbert, Ayuko Ota-Setlik, Krystle N. Agans, Amara Luckay, Susan E. Witko, Lena Soukieh, Daniel J. Deer, Chad E. Mire, Heinz Feldmann, Christian Happi, Karla A. Fenton, John H. Eldridge, Thomas W. Geisbert
Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize UDP-GlcNAc. In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-L-norleucine or DON). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cell. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM, and an increased infiltration CD8+ T-cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy resulting in tumor regression and prolonged survival.
Nikita S. Sharma, Vineet K. Gupta, Vanessa T. Garrido, Roey Hadad, Brittany C. Durden, Kousik Kesh, Bhuwan Giri, Anthony Ferrantella, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee
Tyrosine kinase inhibitors (TKIs) induce molecular remission in the majority of patients with chronic myelogenous leukemia (CML), but persistence of CML stem cells hinders cure and necessitates indefinite TKI therapy. We report that CML stem cells upregulate expression of pleiotrophin (PTN) and require cell-autonomous PTN signaling for CML pathogenesis in BCR/ABL+ mice. Constitutive PTN deletion substantially reduced the numbers of CML stem cells capable of initiating CML in vivo. Hematopoietic cell–specific deletion of PTN suppressed CML development in BCR/ABL+ mice, suggesting that cell-autonomous PTN signaling was necessary for CML disease evolution. Mechanistically, PTN promoted CML stem cell survival and TKI resistance via induction of Jun and the unfolded protein response. Human CML cells were also dependent on cell-autonomous PTN signaling and anti–PTN antibody suppressed human CML colony formation and CML repopulation in vivo. Our results suggest that targeted inhibition of PTN has therapeutic potential to eradicate CML stem cells.
Heather A. Himburg, Martina Roos, Tiancheng Fang, Yurun Zhang, Christina M. Termini, Lauren Schlussel, Mindy M. Kim, Amara Pang, Jenny Kan, Liman Zhao, Hyung Suh, Joshua P. Sasine, Gopal Sapparapu, Peter M. Bowers, Gary Schiller, John P. Chute
Polymorphonuclear neutrophils (PMNs) are increasingly recognized to influence solid tumor development, but why their effects are so context-dependent and even frequently divergent remains poorly understood. Using an autochthonous mouse model of uterine cancer and the administration of respiratory hyperoxia as a means to improve tumor oxygenation, we provide in vivo evidence that hypoxia is a potent determinant of tumor-associated PMN phenotypes and direct PMN-tumor cell interactions. Upon relief of tumor hypoxia, PMNs were recruited less intensely to the tumor-bearing uterus but the recruited cells much more effectively killed tumor cells, an activity our data moreover suggested was mediated via their production of NADPH oxidase-derived reactive oxygen species and MMP-9. Simultaneously, their ability to promote tumor cell proliferation, which appeared mediated via their production of neutrophil elastase, was rendered less effective. Relieving tumor hypoxia thus greatly improved net PMN-dependent tumor control, leading to a massive reduction in tumor burden. Remarkably, this outcome was T cell-independent. Together, these findings identify key hypoxia-regulated molecular mechanisms through which PMNs directly induce tumor cell death and proliferation in vivo and suggest that the contrasting properties of PMNs in different tumor settings may in part reflect the effects of hypoxia on direct PMN-tumor cell interactions.
Karim Mahiddine, Adam Blaisdell, Stephany Ma, Amandine Créquer-Grandhomme, Clifford A. Lowell, Adrian Erlebacher
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