Parkinson's disease (PD) is a neurodegenerative disorder associated with loss of striatal dopamine, secondary to degeneration of midbrain dopamine (mDA) neurons in the substantia nigra, rendering cell transplantation a promising therapeutic strategy. To establish human induced pluripotent stem cell (hiPSC)-based autologous cell therapy, we report a platform of core techniques for the production of mDA progenitors as a safe and effective therapeutic product. First, by combining metabolism-regulating microRNAs with reprogramming factors, we developed a method to more efficiently generate clinical grade iPSCs, as evidenced by genomic integrity and unbiased pluripotent potential. Second, we established a “spotting”-based in vitro differentiation methodology to generate functional and healthy mDA cells in a scalable manner. Third, we developed a chemical method that safely eliminates undifferentiated cells from the final product. Dopaminergic cells thus produced express high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features typical of mDA cells. Transplantation of these cells into rodent models of PD robustly restores motor dysfunction and reinnervates host brain, while showing no evidence of tumor formation or redistribution of the implanted cells. We propose that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD.
Bin Song, Young Cha, Sanghyeok Ko, Jeha Jeon, Nayeon Lee, Hyemyung Seo, Kyung-joon Park, In-Hee Lee, Claudia Lopes, Melissa Feitosa, María José Luna, Jin Hyuk Jung, Jisun Kim, Dabin Hwang, Bruce Cohen, Martin Teicher, Pierre Leblanc, Bob Carter, Jeffrey H. Kordower, Vadim Y. Bolshakov, Sek Won Kong, Jeffrey S. Schweitzer, Kwang-Soo Kim
Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed which possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities especially for multi-antibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed globally impacting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dMAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dMAbs cassettes and studied their activity in small and large animals. Sera from animals administered dMAbs neutralized multiple HIV-1 isolates with similar activity to their parental recombinant MAbs. Delivery of multiple dMAbs to a single animal led to increased neutralization breadth. Two dMAbs, PGDM1400 and PGT121, were advanced into non-human primates for study. High peak circulating levels (between 6-34µg/ml) of these dMAbs were measured and the sera of all animals displayed broad neutralizing activity. The dMAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.
Megan C. Wise, Ziyang Xu, Edgar Tello-Ruiz, Charles Beck, Aspen Trautz, Ami Patel, Sarah T.C. Elliott, Neethu Chokkalingam, Sophie Kim, Melissa G. Kerkau, Kar Muthumani, Jingjing Jiang, Paul Fisher, Stephany J. Ramos, Trevor R.F. Smith, Janess Mendoza, Kate E. Broderick, David C. Montefiori, Guido Ferrari, Daniel W. Kulp, Laurent Humeau, David B. Weiner
Background: Chronic HCV-infection is characterized by a severe impairment of HCV-specific CD4 T cell help that is driven by chronic antigen stimulation. We aimed to study the fate of HCV-specific CD4 T cells after viral elimination. Methods:HCV-specific CD4 T cell responses were longitudinally analyzed using MHC class II tetramer-technology, multicolor flow cytometry and RNA sequencing in a cohort of chronically HCV-infected patients undergoing therapy with direct-acting antivirals. In addition, HCV-specific neutralizing antibodies and CXCL13 levels were analyzed. Results: We observed that the frequency of HCV-specific CD4 T cells increased within two weeks after initiation of DAA therapy. Multicolor flow cytometry revealed a downregulation of exhaustion and activation markers and an upregulation of memory-associated markers. While cells with a Th1 phenotype were the predominant subset at baseline, cells with phenotypic and transcriptional characteristics of follicular T helper cells increasingly shaped the circulating HCV-specific CD4 T cell repertoire, suggesting antigen-independent survival of this subset. These changes were accompanied by a decline of HCV-specific neutralizing antibodies and the germinal center activity. Conclusion: We identified a population of HCV-specific CD4 T cells with a follicular T helper cell signature that is maintained after therapy-induced elimination of persistent infection and may constitute an important target population for vaccination efforts to prevent re-infection and immunotherapeutic approaches for persistent viral infections.
Maike Smits, Katharina Zoldan, Naveed Ishaque, Zuguang Gu, Katharina Jechow, Dominik Wieland, Christian Conrad, Roland Eils, Catherine Fauvelle, Thomas F. Baumert, Florian Emmerich, Bertram Bengsch, Christoph Neumann-Haefelin, Maike Hofmann, Robert Thimme, Tobias Boettler
Successful infection by mucosal pathogens requires overcoming the mucus barrier. To better understand this key step, we performed a survey of the interactions between human respiratory mucus and the human pathogen S. pneumoniae. Pneumococcal adherence to adult human nasal fluid was seen only by isolates expressing pilus-1. Robust binding was independent of pilus-1 adhesive properties but required Fab-dependent recognition of RrgB, the pilus shaft protein, by naturally-acquired secretory immunoglobulin A (sIgA). Pilus-1 binding by specific sIgA led to bacterial agglutination, but adherence required interaction of agglutinated pneumococci and entrapment in mucus particles. To test the effect of these interactions in vivo, pneumococci were preincubated with human sIgA prior to intranasal challenge in a mouse model of colonization. sIgA-treatment resulted in rapid immune exclusion of pilus-expressing pneumococci. Our findings predict that immune exclusion would select for non-piliated isolates in individuals who acquired RrgB-specific sIgA from prior episodes of colonization with piliated strains. Accordingly, genomic data comparing isolates carried by mothers and their children showed that mothers are less likely to be colonized with pilus-expressing strains. Our study provides a specific example of immune exclusion involving naturally-acquired antibody in the human host, a major factor driving pneumococcal adaptation.
Ulrike Binsker, John A. Lees, Alexandria J. Hammond, Jeffrey N. Weiser
Glucocorticoids (GCs) are a central component of therapy for patients with T-cell acute lymphoblastic leukemia (T-ALL) and while resistance to GCs is a strong negative prognostic indicator in T-ALL, mechanisms of GC resistance remain poorly understood. Using diagnostic samples from patients enrolled on the frontline Children’s Oncology Group (COG) T-ALL clinical trial AALL1231, we demonstrated that one-third of primary T-ALLs were resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is critical for normal T-cell function and that plays a well-established role in leukemogenesis. We demonstrated that in these T-ALLs and in distinct populations of normal developing thymocytes, GCs paradoxically induced their own resistance by promoting upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augmented downstream signal transduction resulting in increased STAT5 transcriptional output and upregulation of the pro-survival protein BCL-2. Taken together, we demonstrated that IL7 mediates an intrinsic and physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALLs and is reversible with targeted inhibition of the IL7R/JAK/STAT5/BCL-2 axis.
Lauren K. Meyer, Benjamin J. Huang, Cristina Delgado-Martin, Ritu P. Roy, Aaron Hechmer, Anica M. Wandler, Tiffaney L. Vincent, Paolo Fortina, Adam B. Olshen, Brent L. Wood, Terzah M. Horton, Kevin M. Shannon, David T. Teachey, Michelle L. Hermiston
X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) disease is caused by deficiency of the magnesium transporter 1 gene (MAGT1). We studied 23 XMEN patients, 8 of whom were EBV-naïve. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum, and increased CD4-CD8-B220-TCRalpha/beta+ T (abDNT) cells, in addition to the previously described features of an inverted CD4:CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the “Natural-Killer Group 2, member D” (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We investigated XMEN patients and autoimmune lymphoproliferative syndrome (ALPS) patients by deep immunophenotyping (32 immune markers) using Time of Flight Mass Cytometry (CyTOF). Our analysis revealed that the abundance of two populations of naïve B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and normal individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.
Juan C. Ravell, Mami Matsuda-Lennikov, Samuel D. Chauvin, Juan Zou, Matthew Biancalana, Sally J. Deeb, Susan Price, Helen C. Su, Giulia Notarangelo, Ping Jiang, Aaron Morawski, Chrysi Kanellopoulou, Kyle W. Binder, Ratnadeep Mukherjee, James T. Anibal, Brian Sellers, Lixin Zheng, Tingyan He, Alex B. George, Stefania Pittaluga, Astin Powers, David E. Kleiner, Devika Kapuria, Marc Ghany, Sally Hunsberger, Jeffrey I. Cohen, Gulbu Uzel, Jenna Bergerson, Lynne Wolfe, Camilo Toro, William Gahl, Les R. Folio, Helen Matthews, Pam Angelus, Ivan K. Chinn, Jordan S. Orange, Claudia M. Trujillo-Vargas, Jose Luis Franco, Julio Orrego-Arango, Sebastian Gutiérrez-Hincapié, Niraj Chandrakant Patel, Kimiyo Raymond, Turkan Patiroglu, Ekrem Unal, Musa Karakukcu, Alexandre G.R. Day, Pankaj Mehta, Evan Masutani, Suk S. De Ravin, Harry L. Malech, Grégoire Altan-Bonnet, V. Koneti Rao, Matthias Mann, Michael J. Lenardo
Background: Adoptive transfer of donor-derived EBV-specific T-cells (EBV-CTLs) can eradicate EBV associated lymphomas post hematopoietic cell (HCT) or solid organ (SOT) transplants but is not available for most patients. Methods: We developed a 3rd-party, allogeneic, off-the-shelf bank of 330 GMP grade EBV-CTL lines from specifically consented healthy HCT donors. We treated 46 recipients of HCT (N=33) or SOT (N=13) with established EBV associated lymphomas, who failed rituximab therapy, with 3rd-party EBV-CTLs. Treatment cycles consisted of 3 weekly infusions of EBV-CTLs and 3 weeks of observation. Results: The EBV-CTLs did not induce significant toxicities or graft injury. One patient developed grade I skin GVHD requiring topical therapy. Complete and sustained partial remissions were achieved in 68% of HCT recipients and 54% of SOT recipients. For patients who achieved CR/PR or stable disease after cycle 1, overall survival was 88.9% and 81.8% respectively at 1 year. Although only 1/11 patients (9.1%) with progression of disease (POD) after cycle 1 who received additional EBV-CTLs from the same donor survived, 3 of 5 with POD subsequently treated with EBV-CTLs from a different donor achieved CR or durable PR (60%) and survive > 1 year. Maximal responses were achieved after a median of 2 cycles. Conclusions: Third party EBV-CTLs of defined HLA restriction provide safe, immediately accessible treatment for EBV PTLD. Secondary treatment with EBV-CTLs restricted by a different HLA allele (switch therapy) can also induce remissions if initial EBV-CTLs are ineffective. These results suggest a promising potential therapy for patients with rituximab refractory EBV-associated lymphoma post transplant. Phase II protocols (NCT01498484 and NCT00002663) were approved by the Institutional Review Board at Memorial Sloan Kettering Cancer Center, Food and Drug Administration and National Marrow Donor Program. This work was supported through NIH grants CA23766, NIH R21CA162002, Aubrey Fund, The Claire Tow Foundation, Major Family Foundation, Max Cure Foundation, Richard “Rick” J. EIsemann Pediatric Research Fund, Banbury Foundation, Edith Robertson Foundation, Larry Smead Foundation. In June 2015 Atara Biotherapeutics licensed the EBV-CTL bank and is developing this as ATA-129.
Susan Prockop, Ekaterina Doubrovina, Stephanie Suser, Glenn Heller, Juliet Barker, Parastoo Dahi, Miguel A. Perales, Esperanza Papadopoulos, Craig Sauter, Hugo Castro-Malaspina, Farid Boulad, Kevin J. Curran, Sergio Giralt, Boglarka Gyurkocza, Katharine C. Hsu, Ann Jakubowski, Alan M. Hanash, Nancy A. Kernan, Rachel Kobos, Guenther Koehne, Heather Landau, Doris Ponce, Barbara Spitzer, James W. Young, Gerald Behr, Mark Dunphy, Sofia Haque, Julie Teruya-Feldstein, Maria Arcila, Christine Moung, Susan Hsu, Aisha Hasan, Richard J. O'Reilly
Leptomeningeal anastomoses or pial collateral vessels play a critical role in cerebral blood flow (CBF) restoration following ischemic stroke. The magnitude of this adaptive response is postulated to be controlled by the endothelium, although the underlying molecular mechanisms remain under investigation. Here we demonstrated that endothelial genetic deletion, using EphA4f/f/Tie2-Cre and EphA4f/f/VeCahderin-CreERT2 mice and vessel painting strategies, implicated EphA4 receptor tyrosine kinase as a major suppressor of pial collateral remodeling, CBF and functional recovery following permanent middle cerebral artery occlusion. Pial collateral remodeling is limited by the cross talk between EphA4-Tie2 signaling in vascular endothelial cells, which is mediated through p-Akt regulation. Furthermore, peptide inhibition of EphA4 resulted in acceleration of the pial arteriogenic response. Our findings demonstrate EphA4 is a negative regulator of Tie2 receptor signaling which limits pial collateral arteriogenesis following cerebrovascular occlusion. Therapeutic targeting of EphA4 and/or Tie2 represents an attractive new strategy for improving collateral function, neural tissue health and functional recovery following ischemic stroke.
Benjamin Okyere, William A. Mills III, Xia Wang, Michael Chen, Jiang Chen, Amanda Hazy, Yun Qian, John B. Matson, Michelle H. Theus
Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1-ENG-Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K-Akt-mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine-kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs—including in HHT patient blood outgrowth ECs—and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in HHT patients.
Santiago Ruiz, Haitian Zhao, Pallavi Chandakkar, Julien Papoin, Hyunwoo Choi, Aya Nomura-Kitabayashi, Radhika Patel, Matthew Gillen, Li Diao, Prodyot K. Chatterjee, Mingzhu He, Yousef Al-Abed, Ping Wang, Christine N. Metz, S. Paul Oh, Lionel Blanc, Fabien Campagne, Philippe Marambaud
EGFR mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (pSTAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a pSTAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib and osimertinib respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib treated patients after disease progression.
Emilie Bousquet Mur, Sara Bernardo, Laura Papon, Maicol Mancini, Eric Fabbrizio, Marion Goussard, Irene Ferrer, Anais Giry, Xavier Quantin, Jean-Louis Pujol, Olivier Calvayrac, Herwig P. Moll, Yaël Glasson, Nelly Pirot, Andrei Turtoi, Marta Cañamero, Kwok-Kin Wong, Yosef Yarden, Emilio Casanova, Jean-Charles Soria, Jacques Colinge, Christian W. Siebel, Julien Mazieres, Gilles Favre, Luis Paz-Ares, Antonio Maraver
The corneocyte lipid envelope composed of covalently bound ceramides and fatty acids is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7 knockout model we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7-/- epidermis, quantitative LC-MS assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its non-enzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.
Takuya Takeichi, Tetsuya Hirabayashi, Yuki Miyasaka, Akane Kawamoto, Yusuke Okuno, Shijima Taguchi, Kana Tanahashi, Chiaki Murase, Hiroyuki Takama, Kosei Tanaka, William E. Boeglin, M. Wade Calcutt, Daisuke Watanabe, Michihiro Kono, Yoshinao Muro, Junko Ishikawa, Tamio Ohno, Alan R. Brash, Masashi Akiyama
The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily-life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low angle X-ray diffraction and super-resolution microscopy revealed that the impaired muscle relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling, binding- and contractility assays with recombinant KBTBD13, novel Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models and a GFP-labeled Kbtbd13- transgenic zebrafish model we discovered that KBTBD13 binds to actin – a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.
Josine M. de Winter, Joery P. Molenaar, Michaela Yuen, Robbert van der Pijl, Shengyi Shen, Stefan Conijn, Martijn van de Locht, Menne Willigenburg, Sylvia J.P. Bogaards, Esmee S.B. van Kleef, Saskia Lassche, Malin Persson, Dilson E. Rassier, Tamar E. Sztal, Avnika A. Ruparelia, Viola Oorschot, Georg Ramm, Thomas E. Hall, Zherui Xiong, Christopher N. Johnson, Frank Li, Balazs Kiss, Noelia Lozano-Vidal, Reinier A. Boon, Manuela Marabita, Leonardo Nogara, Bert Blaauw, Richard J. Rodenburg, Benno Kϋsters, Jonne Doorduin, Alan H. Beggs, Henk Granzier, Ken Campbell, Weikang Ma, Thomas Irving, Edoardo Malfatti, Norma B. Romero, Robert J. Bryson-Richardson, Baziel G.M. van Engelen, Nicol C. Voermans, Coen A.C. Ottenheijm
Immunotherapy targeting programmed cell death-1 (PD-1) induces durable antitumor efficacy in many types of cancer. However, such clinical benefit is limited because of the insufficient reinvigoration of antitumor immunity with the drug alone; therefore, rational therapeutic combinations are required to improve its efficacy. In our preclinical study, we evaluated the antitumor effect of U3-1402, a human epidermal growth factor receptor 3 (HER3)–targeting antibody-drug conjugate, and its potential synergism with PD-1 inhibition. Using a syngeneic mouse tumor model that is refractory to anti–PD-1 therapy, treatment with U3-1402 exhibited an obvious antitumor effect via direct lysis of tumor cells. Disruption of tumor cells by U3-1402 enhanced the infiltration of innate and adaptive immune cells. Chemotherapy with exatecan derivative (Dxd: the drug payload of U3-1402) revealed that the enhanced antitumor immunity produced by U3-1402 was associated with the induction of alarmins including HMGB-1 via tumor-specific cytotoxicity. Notably, U3-1402 significantly sensitized the tumor to PD-1 blockade, as a combination of U3-1402 and the PD-1 inhibitor significantly enhanced antitumor immunity. Further, clinical analyses indicated that tumor-specific HER3 expression was frequently observed in patients with PD-1 inhibitor–resistant solid tumors. Overall, U3-1402 is a promising candidate as a partner of immunotherapy for such patients.
Koji Haratani, Kimio Yonesaka, Shiki Takamura, Osamu Maenishi, Ryoji Kato, Naoki Takegawa, Hisato Kawakami, Kaoru Tanaka, Hidetoshi Hayashi, Masayuki Takeda, Naoyuki Maeda, Takashi Kagari, Kenji Hirotani, Junji Tsurutani, Kazuto Nishio, Katsumi Doi, Masaaki Miyazawa, Kazuhiko Nakagawa
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
Tuberculosis (TB) remains a major infectious disease worldwide. TB treatment displays a bi-phasic bacterial clearance, in which the majority of bacteria clear within the first month of treatment, but residual bacteria remains non-responsive to treatment and eventually may become resistant. Here, we have shown that Mycobacterium tuberculosis (M.tb) is taken up by mesenchymal stem cells (MSCs), where it established dormancy and became highly non-responsive to isoniazid, a major constituent of Directly Observed Treatment Short-course (DOTS). Dormant M.tb induced quiescence in MSCs and promoted their long-term survival. Unlike macrophages, where M.tb resides in early-phagosomal compartments, in MSCs the majority of bacilli were found in the cytosol, where they promoted rapid lipid-synthesis, hiding within lipid-droplets. Inhibition of lipid-synthesis prevented dormancy and sensitized the organisms to isoniazid. Thus, we have established that M.tb gains dormancy in MSCs, which thus serve as a long-term natural-reservoir of dormant M.tb. Interestingly, in the murine-model of TB, induction of autophagy eliminated M.tb from MSCs and consequently, the addition of rapamycin to an isoniazid treatment regimen successfully attained sterile clearance and prevented disease reactivation.
Samreen Fatima, Shashank Shivaji Kamble, Ved Prakash Dwivedi, Debapriya Bhattacharya, Santosh Kumar, Anand Ranganathan, Luc Van Kaer, Sujata Mohanty, Gobardhan Das
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