Methods in Molecular Biology 1190 Gaetano Vitale Francesca Mion Editors Regulatory B Cells Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Regulatory B Cells Methods and Protocols Edited by Gaetano V itale and Francesca Mion Department of Medical and Biological Sciences, University of Udine, Udine, Italy Editors Gaetano Vitale Francesca Mion Department of Medical and Biological Sciences Department of Medical and Biological Sciences University of Udine University of Udine Udine, Italy Udine, Italy ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1160-8 ISBN 978-1-4939-1161-5 (eBook) DOI 10.1007/978-1-4939-1161-5 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014942972 © Springer Science+Business Media New York 2 014 This work is subject to copyright. 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Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Pre face The regulation of the immune responses is a rapidly growing and fascinating experimental fi eld that in the last two decades has attracted the interest of an increasing number of labo- ratories. An emerging concept in the fi eld of immune regulation is that immune homeostasis is maintained as a result of the cooperation of many cell types that have both effector and regulatory functions. Both innate and adaptive immune recognition mechanisms contribute to the optimal induction of multiple cell types that interact to produce effector and regula- tory functions. Immune regulation is obtained by tolerance versus self and harmless antigens and by control of immune response amplifi cation. Tolerance prevents lymphocyte activation whereas control restrains the activity of effector cells. Tolerance and control operate by means of cell-intrinsic and cell-extrinsic mechanisms. In particular, cell-extrinsic immune regulation depends upon the activity of several types of immune-suppressive cells often indicated as regulatory cells. B cells are new entrees in the fi eld of immune regulation despite evidence of their role in immune suppression dating back in the 1970s. Nevertheless, numerous recent studies have established their essential role in the control of immune-mediated infl ammation and tolerance. The regulatory function of B cells has been related to their ability to produce the anti-infl ammatory cytokine IL-10. IL-10 secretion is not the only regulatory mechanism operated by B cells: regulatory B cells (Breg) are able to induce regulatory T cells and other suppressive cell types both by contact-mediated interactions and by the production of secreted factors. Many populations of B cells with different phenotypic markers and devel- opmental origin have been recognized to exert a regulatory suppressive function in several immunological settings, including infections, infl ammation, and autoimmunity. The investigation of the mechanisms of immune regulation requires many techniques applied to different cell types. Also, there is an emerging interest in the regulatory role of B cells in many different pathological settings, both in the mouse and in humans. The aim of this book is to present to immunologists as well as cell and molecular biologists interested in B cell biology the current experimental setups and methodologies useful for the identifi cation and characterization of B cells with suppressive functions and for the study of their biological and immunological properties. The book should be of interest to many labs that focus on the study of the regulatory function of B cells as well as to those labs that would like to study the possible function of this cell type in the pathophysiology of a given disease. The book is organized into four parts: the fi rst part contains the basic methods for the isolation and immunophenotypical characterization of specifi c B cell subsets from mouse and human tissues; the second part presents methods useful for the investigation of the mechanisms of immune suppression operated by B cells; the third part presents several experimental approaches for the ex vivo generation/expansion of IL-10-producing B cells; and fi nally, the fourth part presents procedures useful for the study of the immune suppres- sive function of B cells in specifi c pathological settings. Udine, Italy Gaetano V itale Francesca M ion v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x PART I PURIFICATION AND PHENOTYPIC CHARACTERIZATION OF B CELL SUBSETS 1 Purification and Immunophenotypic Characterization of Murine MZ and T2-MZP Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 M. M anuela R osado, M arco S carsella, S imona C ascioli, Ezio Giorda, and Rita C arsetti 2 P urification and Immune Phenotyping of B-1 Cells from Body Cavities of Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 Vanessa Y enson and Nicole B aumgarth 3 P urification and Immunophenotypic Characterization of Murine B10 B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5 Chao Hong and X iao-Ming Gao 4 Purification and Immunophenotypic Characterization of Human B Cells with Regulatory Functions. . . . . . . . . . . . . . . . . . . . . . . . . 45 Adèle de M asson, H élène Le B uanec, and J ean-David B ouaziz PART II MECHANISMS OF IMMUNE SUPPRESSION BY B CELLS 5 IL-10 Detection in Murine B Cells: Pros and Cons of the Different Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Xiang L in, Jun D eng, and Liwei Lu 6 T GF-β Detection and Measurement in Murine B Cells: Pros and Cons of the Different Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 Yoshiyuki M ishima, S hunji I shihara, J onathan J. H ansen, and Y oshikazu Kinoshita 7 Characterization and Activity of Fas Ligand Producing CD5+ B Cells . . . . . . . 8 1 Steven K. Lundy and Matthew W . K linker PART III METHODS FOR THE EX VIVO GENERATION/EXPANSION OF IL-10- PRODUCING B CELLS 8 Utilization of a Lentiviral System for the Generation of B Cells with Regulatory Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 05 Elisabeth C alderón-Gómez and Simon F illatreau 9 E x Vivo Generation of Murine IL-10-Producing B Cells by Fusokines. . . . . . . 1 15 Aurélie T ormo, J iusheng D eng, E douard Al-Chami, Sonia Ziouani, and Moutih R afei vii viii Contents 10 Toll-Like Receptor Ligation for the Induction of Regulatory B Cells. . . . . . . . 1 27 Luciën E . P. M . v an der Vlugt, S imone H aeberlein, Wouter de Graaf, T yrill E. D . M artha, and H ermelijn H. Smits 11 T he Generation of Regulatory B Cells by Helminth Parasites . . . . . . . . . . . . . 1 43 Adnan R. Khan, S ylvie A mu, S ean P . S aunders, and Padraic G . Fallon 12 U se of Cocultures for the Study of Cellular Interactions Influencing B Cell Regulatory Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Francesca Mion, F ederica B envenuti, C arlo P ucillo, and Gaetano V itale PART IV STUDY OF REGULATORY B CELLS IN PATHOLOGICAL SETTINGS 13 R egulatory B Cells in Experimental Mouse Models of Arthritis. . . . . . . . . . . . 1 83 Elizabeth C . Rosser and Claudia M auri 14 Regulatory B Cells in Mouse Models of Systemic Lupus Erythematosus (SLE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 95 Takashi M atsushita 15 Regulatory B Cells in Allergic Airways Disease and Asthma. . . . . . . . . . . . . . . 2 07 Prabitha Natarajan, L inda A. G uernsey, and C raig M. Schramm 16 Regulatory B Cells in Mouse Models of Intestinal Inflammation. . . . . . . . . . . 2 27 Atsushi N ishida, C indy W . L au, E miko M izoguchi, and A tsushi M izoguchi 17 R egulatory B Cells in Experimental Autoimmune Encephalomyelitis (EAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 43 Avijit Ray and Sreemanti B asu 18 Regulatory B Cells, Helminths, and Multiple Sclerosis. . . . . . . . . . . . . . . . . . . 2 57 Jorge Correale and Tomas Rivero E quiza 19 G eneration and Identification of Tumor-Evoked Regulatory B Cells . . . . . . . . 2 71 Arya B iragyn, C atalina Lee-Chang, and M onica Bodogai Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 91 Contributors EDOUARD AL-CHAMI • The Department of Pharmacology , Université de Montréal , Montreal , Q C , Canada SYLVIE A MU • Institute of Molecular Medicine, St James’s Hospital , D ublin, I reland SREEMANTI B ASU • BloodCenter of Wisconsin, Blood Research Institute , Milwaukee, WI , U SA NICOLE B AUMGARTH • Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California , Davis, CA , U SA ; D epartment of Pathology, Microbiology, and Immunology, School of Veterinary Medicine , University of California , Davis, CA , U SA FEDERICA BENVENUTI • International Centre for Genetic Engineering and Biotechnology , Trieste, I taly ARYA B IRAGYN • Immunoregulation Section, N ational Institute on Aging, National Institutes of Health , B altimore, MD, USA; Laboratory of Molecular Biology and Immunology, Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA MONICA BODOGAI • Immunoregulation Section, Laboratory of Molecular Biology and Immunology , National Institute on Aging, National Institutes of Health , B altimore , MD, USA JEAN-DAVID BOUAZIZ • INSERM U976 , Paris, F rance ; U niversité Paris Diderot, Sorbonne Paris Cité , Paris, France ; L aboratory of Immunology, Dermatology & Oncology, UMR-S 976 , P aris, France ; Service de Dermatologie, Hôpital Saint Louis , Paris, F rance HÉLÈNE LE BUANEC • INSERM U976 , P aris , F rance ; U niversité Paris Diderot, Sorbonne Paris Cité , Paris, France ; L aboratory of Immunology, Dermatology & Oncology, UMR-S 976 , P aris, France ; Service de Dermatologie, Hôpital Saint Louis , Paris, F rance ELISABETH CALDERÓN-GÓMEZ • Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) , B arcelona, S pain RITA CARSETTI • Immunology Unit, Research Center Ospedale Pediatrico Bambino Gesù, IRCSS, Rome, Italy; Clinical Laboratories, Research Center Ospedale Pediatrico Bambino Gesù, IRCSS, Rome Italy SIMONA CASCIOLI • Immunology Unit, Research Center Ospedale Pediatrico Bambino Gesù, IRCSS , R ome , I taly JORGE CORREALE • Department of Neurology, Institute for Neurological Research Dr. Raúl Carrea, FLENI , B uenos Aires, Argentina JUN DENG • Department of Pathology, T he University of Hong Kong , H ong Kong, Hong Kong JIUSHENG DENG • Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University , Atlanta , G A , USA TOMAS R IVERO EQUIZA • Department of Neurology, I nstitute for Neurological Research Dr. Raúl Carrea, FLENI , B uenos Aires, Argentina PADRAIC G . F ALLON • School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin , Dublin, I reland ; I nstitute of Molecular Medicine, St James’s Hospital , Dublin, I reland; National Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland ix