HIV Reservoirs: Methods and Protocols (Methods in Molecular Biology, 2407) 1071618709, 9781071618707

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Methods in Molecular Biology 2407

Guido Poli Elisa Vicenzi Fabio Romerio Editors

HIV Reservoirs Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

HIV Reservoirs Methods and Protocols

Edited by

Guido Poli School of Medicine and Human Immuno-Virology (H.I.V.) Group, San Raffaele University and Scientific Institute, Milano, Italy

Elisa Vicenzi Viral Pathogenesis and Biosafety Group, San Raffaele Scientific Institute, Milano, Italy

Fabio Romerio Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Editors Guido Poli School of Medicine and Human Immuno-Virology (H.I.V.) Group San Raffaele University and Scientific Institute Milano, Italy

Elisa Vicenzi Viral Pathogenesis and Biosafety Group San Raffaele Scientific Institute Milano, Italy

Fabio Romerio Department of Molecular and Comparative Pathobiology Johns Hopkins University School of Medicine Baltimore, MD, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-1870-7 ISBN 978-1-0716-1871-4 (eBook) https://doi.org/10.1007/978-1-0716-1871-4 © Springer Science+Business Media, LLC, part of Springer Nature 2022, Corrected Publication 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface In the early 1980s, a new deadly disease struck the world. Initially it affected gay communities in the USA, but soon it spread to other risk groups and emerged worldwide. Since its main clinical features were associated with a profound immunological depression occurring in people with an otherwise healthy immune system, the new pathology was quickly called as “Acquired Immuno-Deficiency Syndrome (AIDS),” an operational definition that has never been amended thereafter. The etiologic agent causing AIDS was soon identified in a previously unknown virus belonging to the retroviridae, shortly after named “Human Immunodeficiency Virus (HIV).” As all retroviruses, HIV integrates the DNA version of its genome into host cell chromosomes and in such form (termed “provirus”) persists indefinitely. Although most infected cells die as a consequence of the cytopathic effects of HIV or because of a related immune response, some of them survive, return to a quiescent state in which the virus lies dormant (proviral latency), and persist indefinitely as long as the cells live forming what is nowadays commonly referred to as the “viral reservoir (VR).” As immune cells, most notably resting memory CD4+ T cells, undergo homeostatic proliferation, the VR persists for the entire life of the individual even under fully suppressive antiretroviral therapy. The relevance of the VR has been paradoxically highlighted by the great success of combination antiretroviral therapy (cART) that was introduced in clinical practice in the mid-1990s by combining pharmacologic inhibitors of two viral enzymes: reverse transcriptase (that converts the viral genomic RNA into a DNA version capable of integrating into the host cell DNA) and protease (required to process the p55 Gag polyprotein following viral budding to obtain mature, infectious virions). Nowadays, cART regimens have been supplemented with other classes of antiretroviral drugs including integrase inhibitors and entry/fusion inhibitors, and the vast majority of treated patients observe the disappearance of detectable virus in their peripheral blood (viremia), thus becoming non-contagious to others (U¼U, i.e., undetectable equals untransmissible). Nonetheless, cART interruption almost invariably results in the rapid resurgence of viremia and disease progression if therapy is not quickly restarted. This rapid viral rebound is mostly, if not exclusively, caused by the presence of the VR, although only a minor fraction (ca. 10%) of the persistently infected cells harbors replication-competent proviruses, whereas the remainder carry defective proviruses of unclear clinical relevance. This absolute limitation of cART has fueled a new research field aimed at the understanding of the cellular composition of the VR, the development of surrogate in vitro and animal models to dissect out its molecular determinants, the discovery of more sophisticated methods of quantification of the VR, and the identification of pharmacological and biologic agents that could either eradicate or permanently silence the integrated genome of replication-competent proviruses. The aim of this book is to summarize the intense efforts of the international scientific community toward the development of methods and models to study the VR with the ultimate goal of achieving a “Functional Cure” of HIV infection. Part I will describe the most relevant cell lines that have represented the earliest models to investigate proviral latency and reactivation and that are still being used as convenient models to identify novel potential determinants of viral latency. Next, the book will describe in vitro and ex vivo primary cell models (Part II) and tissue-derived models (Part III) of persistent

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infection that—in comparison to cell lines—more closely resemble the situation in vivo. In vivo models that utilize nonhuman primates or congenitally immune-deficient mice reconstituted with human immune cells are presented in Part IV, and methods for the detection and analysis of the reservoir will be explored in Part V. Finally, Part VI focuses on the current approaches to achieve either a “Functional Cure” or at least cART-free long-term remission; these include therapeutic latency reversal, durable proviral silencing, and genome editing approaches. Our ambition is not only to offer the reader a comprehensive, updated collection of state-of-the-art methodologies and models to tackle the VR but also to entice them in this fascinating “last battle” against one of the most formidable killers of mankind: the Human Immunodeficiency Virus. Milano, Italy Milano, Italy Baltimore, MD, USA

Guido Poli Elisa Vicenzi Fabio Romerio

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

CELL LINES WITH MINIMAL BUT INDUCIBLE PROVIRAL EXPRESSION

1 Jurkat-Derived (J-Lat, J1.1, and Jurkat E4) and CEM-Derived T Cell Lines (8E5 and ACH-2) as Models of Reversible Proviral Latency. . . . . . . . . . . . . . . . . . Anthony Rodari, Guido Poli, and Carine Van Lint 2 U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible Proviral Latency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Guido Poli

PART II

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IN VITRO AND EX VIVO PRIMARY CELL MODELS OF MINIMAL BUT INDUCIBLE PROVIRAL EXPRESSION

3 An In Vitro System to Model the Establishment and Reactivation of HIV-1 Latency in Primary Human CD4+ T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Rui Li and Fabio Romerio 4 The Cultured TCM Model of HIV Latency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Alberto Bosque 5 A Reliable Primary Cell Model for HIV Latency: The QUECEL (Quiescent Effector Cell Latency) Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Meenakshi Shukla, Fredrick Kizito, Uri Mbonye, Kien Nguyen, Curtis Dobrowolski, and Jonathan Karn 6 TGF-β Signaling Supports HIV Latency in a Memory CD4+ T Cell Based In Vitro Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Sydney Bergstresser and Deanna A. Kulpa 7 Flow Cytometry Sorting of Memory CCR6+CD4+ T-Cells for HIV Reservoir Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Ame´lie Cattin, Augustine Fert, Delphine Planas, and Petronela Ancuta 8 Human Monocyte-Derived Macrophages (MDM): Model 1 (GM-CSF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Claudia Alteri, Lorenzo Piermatteo, Francesca Ceccherini Silberstein, Valentina Svicher, and Carlo Federico Perno 9 Human Monocyte-Derived Macrophages (MDM): Model 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Francesca Graziano, Elisa Vicenzi, and Guido Poli 10 Modeling HIV Latency in Astrocytes with the Human Neural Progenitor Cell Line HNSC.100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Amelie Bauer and Ruth Brack-Werner

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Hematopoietic Stem and Progenitor Cells (HSPCs) . . . . . . . . . . . . . . . . . . . . . . . . 115 Valeri H. Terry, Gretchen E. Zimmerman, Maria C. Virgilio, Mark M. Painter, Dale Bixby, and Kathleen L. Collins

PART III 12 13

IN VITRO AND EX VIVO TISSUE-DERIVED MODELS OF REVERSIBLE PROVIRAL LATENCY

Ex Vivo HIV Infection Model of Cervico-Vaginal and Rectal Tissue . . . . . . . . . . 157 Louise A. Ouattara, Nikolas C. Vann, and Gustavo F. Doncel More than a Gender Issue: Testis as a Distinctive HIV Reservoir and Its Implication for Viral Eradication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Jean-Pierre Routy, Franck P. Dupuy, John Lin, and Ste´phane Isnard

PART IV ANIMAL MODELS 14

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Experimental Models to Study HIV Latency Reversal from Male Genital Myeloid Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fernando Real, Yonatan Ganor, and Morgane Bomsel Decidua Basalis: An Ex Vivo Model to Study HIV-1 Infection During Pregnancy and Beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nabila Jabrane-Ferrat and Hicham El Costa Designing Cure Studies in NHPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amir Dashti, Vidisha Singh, and Ann Chahroudi Human Hematopoietic Stem Cell (HSC)-Engrafted NSG Mice for HIV Latency Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Triana Rivera-Megias, Nhut M. Le, and Alonso Heredia Inducing Long-Term HIV-1 Latency in the TKO-BLT Mouse Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yunyun Di and Kerry J. Lavender

PART V 19

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METHODS FOR DETECTION AND ANALYSIS OF THE RESERVOIR

In Situ Multiplexing to Identify, Quantify, and Phenotype the HIV-1/SIV Reservoir Within Lymphoid Tissue. . . . . . . . . . . . . . . . . . . . . . . . . Kathleen Busman-Sahay, Michael D. Nekorchuk, Carly Elizabeth Starke, Chi Ngai Chan, and Jacob D. Estes Single-Cell Multiparametric Analysis of Rare HIV-Infected Cells Identified by Duplexed RNAflow-FISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mathieu Dube´ and Daniel E. Kaufmann Ex Vivo Differentiation of Resting CD4+ T Lymphocytes Enhances Detection of Replication Competent HIV-1 in Viral Outgrowth Assays . . . . . . . . . . . . . . . . . Elizabeth R. Wonderlich, Monica D. Reece, and Deanna A. Kulpa Quantitative Viral Outgrowth Assay to Measure the Functional SIV Reservoir in Myeloid Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. M. Abreu, R. T. Veenhuis, E. N. Shirk, S. E. Queen, B. T. Bullock, J. L. Mankowski, L. Gama, and J. E. Clements

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Near-Full-Length Single-Genome HIV-1 DNA Sequencing . . . . . . . . . . . . . . . . . 357 Guinevere Q. Lee and Mathias Lichterfeld TILDA: Tat/Rev Induced Limiting Dilution Assay . . . . . . . . . . . . . . . . . . . . . . . . . 365 Cynthia Lungu and Francesco A. Procopio

PART VI

CURE AND LONG-TERM REMISSION STRATEGIES

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Latency Reversal and Clearance of Persistent HIV Infection. . . . . . . . . . . . . . . . . . 375 David M. Margolis 26 Cure and Long-Term Remission Strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Luisa Mori and Susana T. Valente 27 Pathways Toward a Functional HIV-1 Cure: Balancing Promise and Perils of CRISPR Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429 Jonathan Herskovitz, Mahmudul Hasan, Milankumar Patel, Bhavesh D. Kevadiya, and Howard E. Gendelman Correction to: Designing Cure Studies in NHPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C1 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors C. M. ABREU • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA CLAUDIA ALTERI • Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy; Multimodal Medicine Research Area, Bambino Gesu´ Children0 s Hospital, IRCCS, Rome, Italy PETRONELA ANCUTA • De´partement de microbiologie, infectiologie et immunologie, Faculte´ de me´decine, Universite´ de Montre´al, Montre´al, QC, Canada; CHUM-Research Centre, Montre´al, QC, Canada AMELIE BAUER • Institute of Virology, Helmholtz Zentrum Mu¨nchen-Deutsches Forschungszentrum fu¨r Umwelt und Gesundheit, Neuherberg, Germany SYDNEY BERGSTRESSER • Department of Pathology and Laboratory Medicine, Emory University School of Medicine, and Yerkes National Primate Research Center, Atlanta, GA, USA DALE BIXBY • Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA MORGANE BOMSEL • Mucosal Entry of HIV and Mucosal Immunity, Institut Cochin, Universite´ de Paris, Paris, France; INSERM U1016, Paris, France; CNRS UMR8104, Paris, France ALBERTO BOSQUE • Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC, USA RUTH BRACK-WERNER • Institute of Virology, Helmholtz Zentrum Mu¨nchen-Deutsches Forschungszentrum fu¨r Umwelt und Gesundheit, Neuherberg, Germany B. T. BULLOCK • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA KATHLEEN BUSMAN-SAHAY • Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, USA AME´LIE CATTIN • De´partement de microbiologie, infectiologie et immunologie, Faculte´ de me´ decine, Universite´ de Montre´al, Montre´al, QC, Canada; CHUM-Research Centre, Montre´ al, QC, Canada ANN CHAHROUDI • Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA CHI NGAI CHAN • Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, USA J. E. CLEMENTS • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA KATHLEEN L. COLLINS • Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA AMIR DASHTI • Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA YUNYUN DI • Department of Biochemistry, Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada

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CURTIS DOBROWOLSKI • Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA GUSTAVO F. DONCEL • CONRAD, Eastern Virginia Medical School, Norfolk, VA, USA MATHIEU DUBE´ • Research Centre of the Centre Hospitalier de l’Universite´ de Montre´al (CRCHUM), Montre´al, QC, Canada FRANCK P. DUPUY • Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montre´al, QC, Canada; Chronic Viral Illness Service, McGill University Health Centre, Montre´al, QC, Canada HICHAM EL COSTA • Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), INSERM-CNRS-University Toulouse III, Toulouse, France JACOB D. ESTES • Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, USA; Division of Pathobiology & Immunology, Oregon National Primate Research Center, Oregon Health & Science University, Portland, OR, USA AUGUSTINE FERT • De´partement de microbiologie, infectiologie et immunologie, Faculte´ de me´ decine, Universite´ de Montre´al, Montre´al, QC, Canada; CHUM-Research Centre, Montre´ al, QC, Canada L. GAMA • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA YONATAN GANOR • Mucosal Entry of HIV and Mucosal Immunity, Institut Cochin, Universite´ de Paris, Paris, France; INSERM U1016, Paris, France; CNRS UMR8104, Paris, France HOWARD E. GENDELMAN • Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA; Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA FRANCESCA GRAZIANO • Translational Sciences, Sanofi, Chilly-Mazarin, France MAHMUDUL HASAN • Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA ALONSO HEREDIA • Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA JONATHAN HERSKOVITZ • Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA STE´PHANE ISNARD • Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, QC, Canada; Chronic Viral Illness Service, McGill University Health Centre, Montreal, QC, Canada; CIHR Canadian HIV Trials Network (CTN), Vancouver, BC, Canada NABILA JABRANE-FERRAT • Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), INSERM-CNRS-University Toulouse III, Toulouse, France JONATHAN KARN • Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, USA DANIEL E. KAUFMANN • Research Centre of the Centre Hospitalier de l’Universite´ de Montre´ al (CRCHUM), Montre´al, QC, Canada; Universite´ de Montre´al, Montre´al, QC, Canada BHAVESH D. KEVADIYA • Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA

Contributors

xiii

FREDRICK KIZITO • Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, USA DEANNA A. KULPA • Department of Pathology and Laboratory Medicine, Emory University School of Medicine, and Yerkes National Primate Research Center, Atlanta, GA, USA KERRY J. LAVENDER • Department of Biochemistry, Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada NHUT M. LE • Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA GUINEVERE Q. LEE • Weill Cornell Medicine, New York, NY, USA RUI LI • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA MATHIAS LICHTERFELD • Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA; Brigham and Women’s Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA JOHN LIN • Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, QC, Canada; Chronic Viral Illness Service, McGill University Health Centre, Montreal, QC, Canada CYNTHIA LUNGU • Department of Viroscience, Erasmus University Medical Center, Rotterdam, Netherlands J. L. MANKOWSKI • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA DAVID M. MARGOLIS • UNC HIV Cure Center, Department of Medicine, and Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA; Department of Epidemiology, University of North Carolina at Chapel Hill School of Public Health, Chapel Hill, NC, USA URI MBONYE • Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, USA LUISA MORI • Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL, USA MICHAEL D. NEKORCHUK • Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, USA KIEN NGUYEN • Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, USA LOUISE A. OUATTARA • CONRAD, Eastern Virginia Medical School, Norfolk, VA, USA MARK M. PAINTER • Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, USA MILANKUMAR PATEL • Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA CARLO FEDERICO PERNO • Saint Camillus International University of Health and Medical Sciences, Rome, Italy; Multimodal Medicine Research Area, Bambino Gesu´ Children0 s Hospital, IRCCS, Rome, Italy LORENZO PIERMATTEO • Department of Experimental Medicine, University of Rome, Rome, Italy DELPHINE PLANAS • De´partement de microbiologie, infectiologie et immunologie, Faculte´ de me´decine, Universite´ de Montre´al, Montre´al, QC, Canada; CHUM-Research Centre, Montre´al, QC, Canada GUIDO POLI • Human Immuno-Virology (H.I.V.) Unit, San Raffaele Scientific Institute and School of Medicine, Vita-Salute San Raffaele University, Milano, Italy

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FRANCESCO A. PROCOPIO • Department of Immunology and Allergy, Lausanne University Hospital, Lausanne, Switzerland S. E. QUEEN • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA FERNANDO REAL • Mucosal Entry of HIV and Mucosal Immunity, Institut Cochin, Universite´ de Paris, Paris, France; INSERM U1016, Paris, France; CNRS UMR8104, Paris, France MONICA D. REECE • Department of Pathology and Laboratory Medicine, Emory University School of Medicine, and Yerkes National Primate Research Center, Atlanta, GA, USA TRIANA RIVERA-MEGIAS • Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA ANTHONY RODARI • Service of Molecular Virology, Department of Molecular Biology (DBM), Universite´ Libre de Bruxelles (ULB), Gosselies, Belgium FABIO ROMERIO • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA JEAN-PIERRE ROUTY • Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, QC, Canada; Chronic Viral Illness Service, McGill University Health Centre, Montreal, QC, Canada; Division of Hematology, McGill University Health Centre, Montreal, QC, Canada E. N. SHIRK • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA MEENAKSHI SHUKLA • Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, USA FRANCESCA CECCHERINI SILBERSTEIN • Department of Experimental Medicine, University of Rome, Rome, Italy VIDISHA SINGH • Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA CARLY ELIZABETH STARKE • Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, USA VALENTINA SVICHER • Department of Experimental Medicine, University of Rome, Rome, Italy VALERI H. TERRY • Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA SUSANA T. VALENTE • Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL, USA CARINE VAN LINT • Service of Molecular Virology, Department of Molecular Biology (DBM), Universite´ Libre de Bruxelles (ULB), Gosselies, Belgium NIKOLAS C. VANN • CONRAD, Eastern Virginia Medical School, Norfolk, VA, USA R. T. VEENHUIS • Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA ELISA VICENZI • Viral Pathogenesis and Biosafety Group, San Raffaele Scientific Institute, Milan, Italy MARIA C. VIRGILIO • Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA; Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA ELIZABETH R. WONDERLICH • Southern Research, Frederick, MD, USA GRETCHEN E. ZIMMERMAN • Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

Part I Cell Lines with Minimal but Inducible Proviral Expression

Chapter 1 Jurkat-Derived (J-Lat, J1.1, and Jurkat E4) and CEM-Derived T Cell Lines (8E5 and ACH-2) as Models of Reversible Proviral Latency Anthony Rodari, Guido Poli, and Carine Van Lint Abstract The introduction of combination antiretroviral therapy (cART) has switched HIV-1 infection from a lethal disease to a chronic one. Indeed, cART is a lifelong treatment since its interruption is always followed by a rapid rebound of viremia from both cellular and anatomical viral reservoirs where the integrated HIV-1 provirus remains transcriptionally silent or maintains low-levels of viral replication, thereby preventing HIV-1 eradication. As therapeutic approach, the “shock and kill” strategy has emerged with the main objective to reactivate HIV-1 transcription from latency by using latency reversing agents (LRAs) prior to kill the reactivated infected cells by improving host immune responses. In this context, the development of tools such as HIV-1 latently infected cell lines have drastically increased our knowledge about HIV-1 latency and how to counteract this highly heterogeneous phenomenon. In this chapter, we will describe several chronically HIV-1 infected T-lymphocytic cell lines as useful surrogate models to study reversible HIV-1 proviral latency in CD4+ T cells in vitro before approaching more complex and expensive models. Key words HIV, Latency, T cell lines, Proviral integration, Virus expression, “Shock and kill”, “Block and lock”

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Introduction Over the past decades, many studies have deciphered the molecular mechanisms underlying viral latency mostly in CD4+ T lymphocytes which mainly occur at the transcriptional level including transcriptional interference, chromatin organization, absence of cellular host factors, and epigenetic modifications [1]. In parallel, to reach an HIV-1 cure, new therapeutic approaches such as “shock and kill” and “block and lock” strategies, have been developed in order to either reactivate or definitively block the viral transcription in latent reservoirs, respectively [2–4]. While the “shock and kill” therapy, applied in combination with cART, aims to reach the goal of an HIV-1 functional cure, the “block and lock” one will free HIV-1 infected patients from cART treatment since its main

Guido Poli et al. (eds.), HIV Reservoirs: Methods and Protocols, Methods in Molecular Biology, vol. 2407, https://doi.org/10.1007/978-1-0716-1871-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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objective is to leave the immune system handling the infection by suppressing any viral reactivation. Because these therapies are mainly targeting viral transcription, their development requires a detailed understanding of HIV-1 transcription and mechanisms of its regulation. In this context, the development of tools such as HIV-1 latently infected cell lines have drastically increased our knowledge about HIV-1 transcription and latency. In addition, such cellular models of HIV-1 infection remain to date a valuable tool together with primary cell models before working with HIV-1-infected patient cells which are less accessible. In this article, we will focus on chronically HIV-1 infected T-lymphocytic cell lines, namely, ACH-2 and 8E5, originated from the infection of CEM cells, and on a group of cell lines derived from Jurkat T cells, namely, J1.1, J-Lat, and Jurkat E4. 1.1 CEM-Derived T Cell Lines (8E5 and ACH-2)

A prominent result of a collaboration between Anthony S. Fauci’s and Malcolm A. Martin’s laboratories at the NIAID in the mid-1980s, and, in particular, of Thomas M. Folks and his research group, was the establishment of the earliest models of cell line harboring integrated proviruses that could be used as convenient models of latent, but inducible HIV expression. Once acutely infected, usually by a CXCR4-using strain such as HIV-1LAI/IIIB (as most cell lines do not express CCR5) T lymphocytic cell lines undergo a variable degree of cytopathicity with formation of syncytia (as a result of the fusogenic properties of gp41 Env) of different size followed by cell death. In particular, Folks and colleagues studied the CD4+ T lymphocytic, CEM-derived A3.01 cell lines [5] and observed that, after the peak of virus replication around day 10 postinfection, a few cells survived that could be expanded in vitro by replenishing the cell culture with fresh RPMI 1640, 10% FCS enriched medium, and subcloned them. Several cell clones showed a prominent downregulation of the CD4 molecule (primary entry receptor for HIV-1) from the cell surface consequent to multiple interactions with HIV-1 gp120/ gp41 Env proteins and harbored integrated proviruses with different degrees of virus production either in unstimulated conditions or after robust cell stimulation. Three basic profiles of HIV-1 expression were observed: 1. “truly latent” cells that contained proviral DNA, but that did not produce virions (as measured by liquid-phase Mg2+-dependent reverse transcriptase, RT, activity or p24 Gag antigen ELISA); 2. latent cells that produce very low to undetectable levels of RT activity or p24 Gag antigen in basal conditions, but promptly responded to extracellular stimuli in terms of a robust viral output 2–3 days poststimulation reaching levels comparable to those observed at the peak of acute viral infection; 3. cells that were spontaneous “viral factories” and did not respond to exogenous stimulation by modifying their profile in terms of virus production. These basic profiles of virus production have been confirmed by recent analysis [6].

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Two of these cell lines, that were originally clonal, 8E5 and ACH-2, became widely used in the research community although for different purposes. 1.1.1 8E5 Cell Line

This cell line was cloned out of A3.01 cells surviving the acute infection by HIV-1LAI/IIIB and showed a detectable production of virions that were, however, defective [7] in that the provirus had a stop codon mutation in the pol gene resulting in the production of RT-negative virus particles [8]. 8E5 cells responded to 5-iodo20 -deoxyuridine (IUdR) in terms of increased expression of viral proteins that could be visualized 24 h after cell stimulation by formation of syncytia when cocultivated with the CD4+ A3.01 T cell line [7]. A single integrated proviral copy was originally described [8] consistently with the clonality of the initial 8E5 cell line [9]. Among other information, 8E5 provided one of the earliest demonstrations that high levels production of viral proteins per se do not cause cytopathicity in the infected cells unlike what observed during acute infection. The peculiar defective feature of the provirus of 8E5 cells have made them suitable as source of positive controls for HIV DNA quantification [10, 11]. In recent years, however, their molecular state has been revised as multiple integration sites and loss of HIV DNA copy numbers have been described [12, 13] without, however, compromising the overall biological features of the cell line.

1.1.2 ACH-2 Cell Line

The origin of ACH-2 is the same of the 8E5 cell line, but, unlike these cells, provided one the earliest models of reversible proviral latency. In particular, virus production from ACH-2 cells was promptly induced upon stimulation with supernatants derived from LPS-stimulated monocyte-derived macrophages (MDM), as tested by RT activity in their culture supernatants, shifting the percentage of cells expressing viral proteins from 10–15% in unstimulated conditions to virtually 100% after 2–3 days as determined by indirect immunofluorescence staining [14]. Biochemical analysis demonstrated that the main, if not only, factor triggering reversal of proviral latency in the majority of ACH-2 cells was the cytokine tumor necrosis factor-α (TNF-α), as subsequent studies confirmed [15]. TNF-α was indeed characterized independently as capable of activating the intracellular transcription factor NF-kB capable of interacting with two (or more) binding sites in the core promoter of HIV-1 provirus [16]. In addition to TNF-α, also phorbol esters such as phorbol 12-myristate 13-acetate (PMA) promptly upregulated HIV-1 expression in ACH-2 cells at least in part by triggering an autocrine loop of endogenously released TNF-α [17]. Later, it was shown that other members of the TNF receptor family, that is, CD30 [18] and OX40 [19], induced high levels of virus production in ACH-2 cells. Similar results with this cell line were observed by heat shock exposure [20], stimulation with heme arginate

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(HA) [21], HMGB1 [22] or CpG oligodeoxynucleotides (CpG ODNs) [23] via activation of the NF-kB pathway. Furthermore, HIV-1 production from ACH-2 cells was also induced by stimulation with pharmacologic inhibitors of intracellular calcium pumps by acting at the transcriptional level, likely via activation of NF-kB [24]. Therefore, ACH-2 cells can be considered a convenient model to investigate natural molecules or pharmacologic agents targeting selectively the NF-kB-dependent pathway of reactivation of HIV proviruses in T lymphocytic cells. In addition, ACH-2, as J-Lat Tat-GFP cell lines later discussed, have been successfully used to test various histone deacetylase (HDAC) inhibitors [25] and of fimepinostat (CUDC-907), an inhibitor of both HDAC and PI3 kinases [26]. Other epigenetic regulators capable of inducing virus production in ACH-2 cells include sodium butyrate [27] whereas amino acid starvation triggered virus production in the HDAC4+ ACH-2 cell line, but not in HDAC negative U1 cells [28]. 1.2 Jurkat-Derived T Cell Lines (J-Lat, J1.1, and Jurkat E4) 1.2.1 J1.1 Cell Line

The Jurkat-derived HIV-1 latently infected cell line J1.1 was generated by the group of Thomas Folks in 1991 [29]. Briefly, Jukat T cells were infected with the CXCR4-dependent (X4) HIV-1LAI/IIIB strain for 2 h at 37  C before being seeded in culture for 2 weeks. Then, the surviving cells were cloned by performing limiting dilutions in 96-well plates at an approximate suspension of 0.1 cell/ well. Next, the growing cultures were assayed for RT activity and the positive ones were expanded and subcloned. Among those RT positive clones, the J1.1 cell line was selected and defined as a new cellular model to study both mechanisms regulating HIV-1 proviral latency and the potential interference of viral infection on T cell function. The phenotypical analysis of this cell line showed that HIV-1 infection caused a significant downregulation of CD3 and CD4 expression from the cell surface resulting in the insensitivity to CD3 stimulation by using anti-CD3 mAb, in terms of proviral reactivation, as measured by p24 Gag antigen release in culture supernatants [29]. In addition, the lack of CD3 expression was correlated to a reduced secretion of interleukin-2 (IL-2) compared to the uninfected parental Jurkat cell line, likely due to a decrease of Ca2+ mobilization. In contrast, p24 Gag production was promptly induced by TNF-α stimulation. Although it was originally assumed that the J1.1 cell line was suitable for studying signaling pathways at the clonal level, a recent study by Symons and colleagues has reported evidence of ongoing virus replication associated with 117 different HIV-1 integration sites per 150,000 cells that remain stable over time [30]. Among these, two major integration sites have been identified, with an equal frequency, located in the deformed epidermal autoregulatory factor 1 (DEAF1) gene and in the chromobox homolog 5 (CBX5), on the chromosome 11 and 12, respectively [30]. Overall, these

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observations suggest that J1.1 cells should be considered as a model of polyclonal, rather than monoclonal, reversible proviral latency. Furthermore, the J1.1 cell line was recently used to set up a new method, called HIV-1 DNA-capture-seq, which characterizes the HIV-1 provirus at a single nucleotide resolution and better defines the HIV-1 integration sites [31]. While this study, on the one hand, confirms that there are two major HIV-1 integration sites in J1.1 cells, the provirus integrated in chromosome 12 harbors a deletion in the 50 -LTR likely causing transcriptional silencing. Altogether, the J1.1 cell line exhibits multiple mechanisms associated with proviral latency, likely reflecting an heterogeneity occurring during infection in vivo. In addition, J1.1 cells offer an interesting model to study the functional interaction between CD3 and CD4, and related molecules, and the cellular pathways involving the CD3 receptor following HIV-1 infection. Recently, J1.1 cells have been used to demonstrate that HIV-1 latency can be reverted by a mechanism involving the formation of a “viral synapse” of cell–cell contact when cocultivated with uninfected Jurkat T cells stimulated by a phorbol myristate acetate (PMA) plus ionomycin combination. This mechanism involved the HIV-1 matrix protein p17 Gag and gp120 Env and the cell surface proteins LFA-1, CD59, and CD81 and HLA class I molecules [32]. 1.2.2 J-Lat Cell Lines

HIV-1 latently infected T-lymphocytic J-Lat cell lines were generated in 2003 by the group of Eric Verdin with the main goal to better characterize postintegration latency mechanisms that affect HIV-1 transcriptional activity [33]. Compared to the previously existing HIV-1 latently infected T-lymphocytic cell lines, already reported since the mid-1980 s [5] and later shown to be characterized by either mutations in the Tat-TAR axis or in the NF-kB binding sites located in the HIV-1 promoter 50 -LTR [34–36], the J-Lat cell lines contained a wild-type HIV-1NL4–3 strain provirus where the nef open reading frame has been replaced by the gene coding for the green fluorescent protein (GFP). Briefly, this cell line was generated by infecting the T-lymphocytic cell line Jurkat with an HIV-1NL4–3 molecular clone depleted of both nef (replaced by the GFP) and env genes. Of note, the depletion of env coding sequence allowed a single round of infection and was complemented by using VSV-G pseudotyped viral particles. Following infection, since GFP expression is under the control of the HIV-1 50 -LTR promoter, the putative HIV-1 latently infected cells corresponded to GFP-negative cells and were sorted by FACS. Then, this remaining pool of cells which was thus composed of both uninfected and latently infected cells were stimulated with TNF-α, known to upregulate proviral expression via activation of the transcription factor NF-κB for which two or more DNA-binding sites are present in the HIV-1 core promoter [37, 38]. The GFP-positive cells, corresponding to latently infected cells that responded to

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TNF-α by reactivating proviral expression, were single-cell sorted and left in culture until they spontaneously returned to a state of proviral latency. By this approach, different J-Lat clones (called 6.3, 8.4, 9.2, 10.6, and 15.4) were generated with the major advantage to be monoclonal and to contain a single copy of a replicationincompetent HIV-1 integrated provirus [33]. Recently, the groups of Sharon R. Lewin and Paul U. Cameron have confirmed the monoclonality of J-Lat clones 8.4, 9.2, 10.6, and 15.4, using next-generation sequencing, and identified the unique integration site of each clone [30]. Of note, as the J-Lat clone 6.3 harbors the same HIV-1 integration site than the J-Lat 9.2 they are considered as “sister clones.” Experimentally, the main interest regarding the incorporation of the GFP gene into the HIV-1 genome is based on its expression which is strictly coordinated with the activation of the HIV-1 50 -LTR promoter and the expression of viral proteins, thereby allowing an immediate and quantitative measurement of viral transcription levels in infected cells by flow cytometry, fluorescence microscopy, or plate-based fluorometry (see detailed protocol of flow cytometry hereafter). Another advantage of flow-cytometric analysis resides in its ability to measure both bulk and single-cell quantification of viral reactivation without any manipulation (such as fixation or staining) that might affect the results while being costeffective. 1.2.3 Jurkat E4 Cell Line

The HIV-1 latently infected cell line Jurkat E4 was generated in 2008 by the group of Jonathan Karn to further understand the epigenetic mechanisms that induce viral latency following HIV-1 infection [39]. This cell line harbors an HIV-1 provirus corresponding to the chimeric molecular infectious clone pNL4.3 [40] in which the d2GFP reporter gene was incorporate to replace the nef coding sequence and to be under the control of the HIV-1 promoter in the 50 -LTR. The strategy that was adopted to select this cell line started by firstly selecting cell clones that were initially expressing the provirus (i.e., they were d2GFP+) before undergoing proviral silencing in the absence of cell stimulation. A unique integration site in this cell line is located in the intronic sequence corresponding to an hypothetical protein called LOC401541 in chromosome 9 [39]. Of interest, this integration site is in an euchromatin environment, similar to what is observed in resting CD4+ T lymphocytes of HIV-1+ individuals [41], thereby reinforcing the interest to use this HIV-1 latently infected cell line to study the effect of latently reversing agents. In addition, FACS analyses have demonstrated that, despite the presence of a wild-type form of Tat, HIV-1 transcriptional activity in Jurkat E4 cells is highly repressed as >95% of the cells were d2GFP. As other similar models, also the provirus harbored in this cell line is easily inducible

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following activation of the NF-κB pathway by TNF-α stimulation. Of note, up to 80% of Jurkat E4 cells were reactivated by TNF-α compared to approximatively 30% of J-Lat 6.3 [33, 39]. In addition, HIV-1 expression in Jurkat E4 cells was also triggered by trichostatin A (TSA), a pan-inhibitor of histone deacetylases (HDAC), suggesting that a proportion of the latency mechanisms affecting the HIV-1 promoter in this cell line is related to epigenetic modifications [39]. 1.3 HIV-1 Transcriptional Reactivation: How to Measure it?

In the literature, many pharmacological agents have already been demonstrated to be potent HIV-1 latency reversing agents (LRAs) [42] but the emergence of new LRAs is currently faster than ever. Thus, the assessment of their reactivation potential will need to be tested on HIV-1 latently infected cell lines as a proof of concept prior to assess their putative effects on HIV-1 latency in a more physiological context of infection corresponding to HIV-1 latently infected primary T cells from HIV+ individuals. Regarding the existing readouts of HIV-1 transcriptional reactivation, we can precisely quantify by performing RT-qPCR assays both HIV-1 initiated and elongated transcripts corresponding to Tar and Tat transcripts, respectively. However, a recent work published by Yukl et al. has demonstrated that the different classes of LRAs are not reversing all transcriptional blocks affecting HIV-1 gene expression [43]. Thus, their method to measure different classes of HIV-1 transcripts has to be considered when deciphering the reactivation potential of a specific LRA. Another common method to measure HIV-1 viral reactivation, following LRA treatment, consists of measuring p24 capsid protein levels in the culture supernatant, which is correlated to HIV-1 viral production. As an alternative method to titer levels of HIV-1 particles in the supernatant, the group of Bruno Verhasselt has set up a novel assay, called SG-PERT, which provide a fast, accurate, and unexpensive method to quantify retroviruses production by measuring the activity of the viral reverse transcriptase (RT) enzyme [44]. In addition, as explained above, the reactivation potential of a specific LRA (alone or in combination with others) is also measurable by performing FACS assays when using HIV-1 latently infected cell lines that have an HIV-1 provirus where the GFP gene has been inserted (J-Lat clones and Jurkat E4). In the next section, we detailed a panel of experiments that can be performed to evaluate the reactivation potential of a specific LRA, alone or in combination.

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Materials LRA Stimulation

1. Complete RPMI medium (85% RPMI 1640 GlutaMAX, 10% fetal bovine serum, 5% penicillin–streptomycin). 2. The LRA of interest should be dissolved in the appropriate solvent (according to manufacturer’s protocol) to obtain a 100 stock solution.

2.2 Well-Established LRA Active on ACH-2 Cells

1. Phorbol esters (PMA, PDBU). PMA1 should be dissolved in either DMSO or Ethanol at 103 to 104 M, and stored at 80  C or 20  C. Immediately prior to use, stock solutions should be diluted in complete medium to obtain a 100 solution (105 M). Then, PMA should be added to cell culture medium at final concentrations ranging from 107 to 109 M. Soluble phorbol esters such as PDBU (Phorbol 12,13dibutyrate) can be used in substitution of PMA at the same final concentrations for experiments aiming at investigating the kinetics of cell stimulation See Note 1. 2. TNF-α (or TNF-α). Cytokine should be diluted in complete medium in order to obtain a 100 stock solution, and aliquots should be stored at 80  C or 20  C. Stock solutions should be thawed immediately before use, and used at final concentrations ranging from 0.1 to 1 ng/mL.

2.3

RT-qPCR Assay

1. Ice-cold phosphate buffer saline (PBS) 1. 2. TRIzol reagent. 3. Chloroform. 4. 50% ethanol–50% isopropanol buffer. 5. 75% ethanol–25% H2O buffer. 6. TURBO DNase kit. 7. Reverse transcription Kit. 8. qPCR Master Mix. 9. Specific oligonucleotide primers designed for amplified HIV-1 RNA transcripts.

2.4

FACS Assay

1. Ice-cold phosphate buffer saline (PBS) 1. 2. Paraformaldehyde 4% (4% paraformaldehyde 96%, PBS 1). 3. FACS buffer (98.8% PBS 1, 0.1% BSA, 0.1% NaN3).

2.5

p24 ELISA

1. HIV-1 p24 ELISA kit.

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Methods ACH-2 Cells

1. For experiments aiming at investigating latency reversal by detection of RT activity or p24 Gag antigen in culture supernatants, cells should be seeded at 1–2  105 cells/mL in complete medium (RPMI 1640, 10% FCS supplemented with glutamine, 2 μmol/mL) in the presence or absence of known stimuli (see below) and allowed to proliferate for 2–4 days without need of replacing the tissue culture medium. 2. For shorter experiments, for example for studying HIV RNAs, cell concentrations of 0.5–1  106 cells. 3. Latency reversal can be typically studied in flat-bottom 96-well plates allowing multiple replicas of the identical condition; it is suggested to design experiments with three or more replicas per single condition. 4. At the desired times after stimulation, 10–20 μL aliquots of cell culture supernatants can be harvested without need of replacement. At the end of the experiment, cells can be harvested from cell suspension, centrifuged, resuspended at the desired cell concentration and volume in order to investigate cellassociated viral and cellular proteins or RNA, as described. Peak levels of viral RNA after cell stimulation are typically observed between 6 and 12 h after cell stimulation; viral proteins accumulate between 24 and 48 h poststimulation.

3.2 LRA Stimulation of Jurkat-Derived Cell Lines

1. The day before the experiment, split the cells to a concentration of 8  105 cells per mL of complete RPMI medium. 2. On the day of the experiment, seed the cells at a specific concentration depending on treatment duration: 106 cells/ mL for 24 h, 0.8  106 cells/mL for 48 h, or 0.6  106 cells/mL for 72 h. Then, stimulate the cells with the LRA of interest (alone or in combination) by adding a specific volume in order to reach the desired final concentration. Of note to optimize the drug concentration, it is important to define a range of concentration that has to be tested in parallel. 3. Collect the cells or cell supernatants after 24 h, 48 h, and 72 h poststimulation and perform the required assay to functionally evaluate the reactivation potential of the compound of interest.

3.3

RNA Extraction

1. At the desired times after stimulation, collect the cells and transfer into a 1.5 mL tube. 2. Centrifugate at 1000  g for 5 min (at room temperature, rt  C) and eliminate the supernatant. 3. Resuspend the pellet in 1 mL of ice-cold PBS 1.

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4. Centrifugate at 1000  g for 5 min (at rt  C) and discard the supernatant. 5. Resuspend the pellet in 1 mL of TRIzol reagent by pipetting. 6. Incubate for 5 to 10 min at rt  C. 7. Add 200 μL of chloroform and homogenize by shaking during 15–30 s. 8. Incubate for 10 min on ice. 9. Centrifugate at 16,000  g for 15 min (at 4  C) and transfer the aqueous phase into a new tube (approximatively 400 μL). 10. Add 500 μL of a 50% ethanol—50% isopropanol buffer and homogenize by vortexing. 11. Incubate overnight at 80  C. 12. Centrifugate at 16,000  g for 15 min (at 4  C) and discard the supernatant. 13. Centrifugate at 16,000  g for 15 min (at 4  C) and discard the remaining supernatant. 14. Rinse the pellet with 1 mL of 75% ethanol buffer and homogenize by inversion. 15. Centrifugate at 9600  g for 15 min (at 4  C) and discard the supernatant. 16. Centrifugate at 16,000  g for 15 min (at 4  C) and discard the remaining supernatant. 17. Incubate on a thermoblock at 55  C for 5–10 min to allow evaporation of the remaining ethanol. 18. Resuspend the pellet in 10–30 μL of RNase-free water depending on pellet size. 19. Incubate for 10 min at 60  C to fully solubilize the RNA pellet in water. 3.4 Turbo DNase Treatment

1. To eliminate DNA contaminants, use a commercial kit to perform a DNase treatment by following the manufacturer’s protocol. 2. Quantify RNA samples using a Nanodrop.

3.5 Reverse Transcription

1. To reverse-transcribe your RNA into a cDNA, use a commercial kit allowing to perform the reverse transcription step by following the manufacturer’s protocol.

3.6 Real-Time Quantitative PCR

1. Under a DNA-free hood, prepare specific qPCR mixes for each type of HIV-1 transcripts containing (per point): (a) x μL of qPCR Master Mix (according with manufacturer’s protocol). (b) 10 nM of reverse primer (final concentration).

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(c) 10 nM of forward primer (final concentration). (d) x μL of water (depending of mix final volume). 2. Transfer x μL of each qPCR mix (according with manufacturer’s protocol) into a 96-well plate. 3. Outside the hood, dilute 10 your cDNA samples in water. 4. Add 5 μL of cDNA (per point) into the 96-well plate containing the x μL of qPCR mix. 5. Seal the 96-well plate. 6. Centrifugate for 1 min at 250  g (at 4  C). 7. Load your 96-well plate into a qPCR machine and check the parameters (depending of the machine brand). 8. Start the run. 3.7

FACS Assay

1. At the desired time after stimulation, collect the cells and transfer into a 1.5 mL tube. 2. Centrifugate at 1200  g for 6 min (at 4  C) and eliminate the supernatant. 3. Resuspend the pellet in 1 mL PBS 1 by pipetting. 4. Repeat steps 2 and 3 once. 5. Centrifugate at 1200  g for 6 min (at 4  C) and discard the supernatant. 6. Resuspend the pellet in 250 μL paraformaldehyde 4%. 7. Incubate for 45 min at 4 C (optional). 8. Add 1 mL PBS 1. 9. Centrifugate at 1200  g for 6 min (at 4  C) and discard the supernatant. 10. Resuspend the pellet in 1 mL PBS 1 by vortexing. 11. Centrifugate at 1200  g for 6 min (at 4  C) and discard the supernatant. 12. Resuspend the pellet in 500 μL FACS buffer and homogenize by pipetting. 13. Transfer into FACS tubes. 14. Measure the fluorescence intensity by using a flow cytometer.

3.8

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p24 ELISA

1. In order to quantify p24 capsid protein in cell culture supernatants, use a specific HIV-1 p24 ELISA kit and follow manufacturer’s protocol.

Notes 1. PMA is a lipophilic compound that enters the cells and cannot be removed by cell centrifugation.

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Acknowledgments CVL acknowledges funding from the Belgian National Fund for Scientific Research (FRS-FNRS, Belgium), the European Union0 s Horizon 2020 research and innovation programme (grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015), the “Fondation Roi Baudouin”, the NEAT (European AIDSTreatment Network) program, the “Fondation Roi Baudouin”, the Internationale Brachet Stiftung (IBS), ViiVHealthcare, the Walloon Region (Fonds de Maturation), “Les Amis des Instituts Pasteur a´ Bruxelles, asbl.”, andthe University of Brussels (ULB Action de Recherche Concerte´e (ARC) grant) related to her work on HIVlatency. The laboratory of CVL is part of the ULB-Cancer Research Centre (U-CRC). AR is a postdoctoral fellow(ULB ARC program). CVL is “Directeur de Recherches” of the F.R.S-FNRS. References 1. Dutilleul A, Rodari A, Van Lint C (2020) Depicting HIV-1 transcriptional mechanisms: a summary of what we know. Viruses 12:1385 2. Abner E, Jordan A (2019) HIV ‘shock and kill’ therapy: in need of revision. Antivir Res 166: 19–34 3. Moranguinho I, Valente ST (2020) Block-andlock: new horizons for a cure for HIV-1. Viruses 12:1443 4. Vansant G, Bruggemans A, Janssens J, Debyser Z (2020) Block-and-lock strategies to cure HIV infection. Viruses 12:84 5. Folks T et al (1985) Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc Natl Acad Sci U S A 82:4539–4543 6. Telwatte S et al (2019) Heterogeneity in HIV and cellular transcription profiles in cell line models of latent and productive infection: implications for HIV latency. Retrovirology 16:32 7. Folks TM et al (1986) Biological and biochemical characterization of a cloned Leu-3- cell surviving infection with the acquired immune deficiency syndrome retrovirus. J Exp Med 164:280–290 8. Lightfoote MM et al (1986) Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol 60:771–775 9. Deichmann M, Bentz M, Haas R (1997) Ultrasensitive FISH is a useful tool for studying

chronic HIV-1 infection. J Virol Methods 65: 19–25 10. Jackson JB et al (1993) Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. ACTG PCR Working Group, and the ACTG PCR Virology Laboratories. J Clin Microbiol 31:3123–3128 11. Bourinbaiar AS, Ajuang-Simbiri K (1996) Simple procedure for estimating the efficiency of PCR. Mol Biotechnol 6:87–89 12. Busby E et al (2017) Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR. Sci Rep 7:1209 13. Wilburn KM et al (2016) Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses. Retrovirology 13:55 14. Clouse KA et al (1989) Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol 142:431–438 15. Folks TM et al (1989) Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci U S A 86:2365–2368 16. Gimble JM et al (1988) Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type 1 is associated with induction of a nuclear factor that binds to the NF-kappa B/core enhancer sequence. J Virol 62:4104–4112

Jurkat-Derived (J-Lat, J1.1, and Jurkat E4) and CEM-Derived T Cell Lines... 17. Poli G et al (1990) Tumor necrosis factor alpha functions in an autocrine manner in the induction of human immunodeficiency virus expression. Proc Natl Acad Sci U S A 87:782–785 18. Biswas P et al (1995) Cross-linking of CD30 induces HIV expression in chronically infected T cells. Immunity 2:587–596 19. Takahashi Y et al (2001) OX40 stimulation by gp34/OX40 ligand enhances productive human immunodeficiency virus type 1 infection. J Virol 75:6748–6757 20. Stanley SK, Bressler PB, Poli G, Fauci AS (1990) Heat shock induction of HIV production from chronically infected promonocytic and T cell lines. J Immunol 145:1120–1126 21. Shankaran P, Vlkova L, Liskova J, Melkova Z (2011) Heme arginate potentiates latent HIV-1 reactivation while inhibiting the acute infection. Antivir Res 92:434–446 22. Thierry S et al (2007) High-mobility group box 1 protein induces HIV-1 expression from persistently infected cells. AIDS 21:283–292 23. Scheller C et al (2004) CpG oligodeoxynucleotides activate HIV replication in latently infected human T cells. J Biol Chem 279: 21897–21902 24. Papp B, Byrn RA (1995) Stimulation of HIV expression by intracellular calcium pump inhibition. J Biol Chem 270:10278–10283 25. Savarino A et al (2009) ‘Shock and kill’ effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence. Retrovirology 6:52 26. Gunst JD et al (2019) Fimepinostat, a novel dual inhibitor of HDAC and PI3K, effectively reverses HIV-1 latency ex vivo without T cell activation. J Virus Erad 5:133–137 27. Laughlin MA, Chang GY, Oakes JW, Gonzalez-Scarano F, Pomerantz RJ (1995) Sodium butyrate stimulation of HIV-1 gene expression: a novel mechanism of induction independent of NF-kappa B. J Acquir Immune Defic Syndr Hum Retrovirol 9:332–339 28. Palmisano I et al (2012) Amino acid starvation induces reactivation of silenced transgenes and latent HIV-1 provirus via down-regulation of histone deacetylase 4 (HDAC4). Proc Natl Acad Sci U S A 109:E2284–E2293 29. Perez VL et al (1991) An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation. J. Immunol 147:3145–3148 30. Symons J et al (2017) HIV integration sites in latently infected cell lines: evidence of ongoing replication. Retrovirology 14:2

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31. Iwase SC et al (2019) HIV-1 DNA-capture-seq is a useful tool for the comprehensive characterization of HIV-1 provirus. Sci Rep 9:12326 32. Okutomi T, Minakawa S, Hirota R, Katagiri K, Morikawa Y (2020) HIV reactivation in latently infected cells with Virological synapselike cell contact. Viruses 12:417 33. Jordan A, Bisgrove D, Verdin E (2003) HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22: 1868–1877 34. Emiliani S et al (1996) A point mutation in the HIV-1 tat responsive element is associated with postintegration latency. Proc Natl Acad Sci U S A 93:6377–6381 35. Emiliani S et al (1998) Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line. J Virol 72:1666–1670 36. Antoni BA, Rabson AB, Kinter A, Bodkin M, Poli G (1994) NF-kappa B-dependent and -independent pathways of HIV activation in a chronically infected T cell line. Virology 202: 684–694 37. Gaynor R (1992) Cellular transcription factors involved in the regulation of HIV-1 gene expression. AIDS 6:347–363 38. Bachu M et al (2012) Multiple NF-κB sites in HIV-1 subtype C long terminal repeat confer superior magnitude of transcription and thereby the enhanced viral predominance. J Biol Chem 287:44714–44735 39. Pearson R et al (2008) Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J Virol 82:12291–12303 40. Bosch V, Pawlita M (1990) Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site. J Virol 64:2337–2344 41. Han Y et al (2004) Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes. J Virol 78:6122–6133 42. Spivak AM, Planelles V (2018) Novel latency reversal agents for HIV-1 cure. Annu Rev Med 69:421–436 43. Yukl SA et al (2018) HIV latency in isolated patient CD4+ T cells may be due to blocks in HIV transcriptional elongation, completion, and splicing. Sci Transl Med 10:eaap9927 44. Vermeire J et al (2012) Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, Lenti- and Retroviral vectors. PLoS One 7:e50859

Chapter 2 U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible Proviral Latency Guido Poli Abstract As already discussed for T cell lines, also myeloid cell lines as served as the earliest models of chronic HIV infection. They were particularly relevant in the late 1980s and early 1990s when most experimental in vitro infections were based on laboratory-adapted “T-cell tropic” strains of HIV-1, such as LAI/IIIB or others, that later were found to rely upon CXCR4 as coreceptor for viral entry in addition to CD4 as primary receptor. Although primary macrophages do express CXCR4 together with CD4, virus replication is much less efficient than that observed with CCR5-using “macrophage-tropic” strains, as discussed separately in this book. Although different myeloid cell lines have been used to generate models of chronic HIV-1 infection that could be used to investigate features of proviral reactivation, as reviewed in (Cassol et al. J Leukoc Biol 80:1018–1030, 2006), two cell lines in particular have been broadly used and will be here discussed: the U937-derived U1 and HL-60–derived OM-10.1. Key words HIV, Latency, Cell line, Myeloid, U937, HL-60, Proviral integration, Virus expression

1 1.1

Introduction U1 Cell Line

As for 8E5 and ACH-2 (see chapter by A. Rodari et al. in this book), Tom Folks’s group in Anthony S. Fauci’s laboratory, in collaboration with Malcolm A. Martin’s laboratory, both at NIAID, NIH, Bethesda, MD (USA), produced a series of chronically infected cell lines, clonal in origin, among which the U1 cell line was particularly characterized as model of reversible proviral latency in a myeloid cell background. U1 cells carry two proviral copies in each cell [1] that are defective in the Tat [2], but bear a functional TAR sequence as demonstrated by the observation that virus production can be rescued in U1 cells (unlike ACH-2 cells) upon either transfection of a Tat-encoding plasmid or incubation with Tat protein [3]. Similar to ACH-2 and other T cell lines, virus production in U1 cells can be reactivated upon cell stimulation with tumor necrosis factor-α/
β (TNF-α/
β) or phorbol esters, such as phorbol-12

Guido Poli et al. (eds.), HIV Reservoirs: Methods and Protocols, Methods in Molecular Biology, vol. 2407, https://doi.org/10.1007/978-1-0716-1871-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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myristate, 13-acetate (PMA) [4], an effect largely mediated by activation of NF-kB-dependent proviral transcription [5]. However, several cytokines and related stimuli proven ineffective on T cell lines like ACH-2, were shown to reverse the state of quasilatency of U1 cells, as reviewed [6]. Among these, Interleukin-1β (IL-1β), Interferon-γ (IFN-γ) and IL-6, this latter characterized by a combined transcriptional and posttranscriptional effect [7]. Of interest, the transcriptional component leading to upregulated proviral transcription induced by IL-6 was later accounted for by the activation of the AP-1 family of transcription factors [8]. A combination of TNF-α and IL-6, IFN-γ, or other cytokines resulted in a synergistic effect on virus reactivation in U1 cells [7]. In parallel to the expansion of the panel of HIV-inductive cytokines beyond TNF-α, also molecules with repressive activities have been characterized in U1 cells. IFN-α was shown to block virion production from both U1 and ACH-2 cell lines stimulated with PMA by a classical “postbudding” effect [9], later shown to be mediated by the upregulation of BST-2/Tetherin [10]. Transforming growth factor-β (TGF-β), a potent immunosuppressive cytokine, as well as retinoic acid (RA), repressed PMA and cytokineinduced reactivation of HIV production in U1 cells, as well as virus replication in primary human monocyte-derived macrophages (MDM) [11]. In this regard, PMA-induced reactivation of proviral HIV expression in U1 cells was shown to be partially sustained by the induction of an autocrine loop involving the release of endogenous TNF-α [12]. Therefore, blocking PMA-induced TNF-α (either in terms of its release or interaction with its cell surface receptor) resulted in ca. 50% reduction of virus output; of interest, complete suppression of PMA-induced reactivation of HIV expression from U1 cells can be achieved by simultaneous blockade of endogenous TNF-α together with cell stimulation with TGF-β or RA. Of note, a second autocrine loop leading to increase HIV-1 expression has been reported for IL-1β [13] whereas IL-4 was later shown to inhibit viral production by a strong enhancement of endogenous IL-1 receptor antagonist (IL-1ra) secretion and a concomitant reduction of IL-1β release [14]. PMA stimulation of U1 cells triggers their differentiation into MDM-like cells, including a dramatic shape change and acquisition of cell adhesion to the plastic substrate. This peculiarity has allowed to exploit PMA-differentiated U1 cells as a model of virion accumulation in subcellular compartments known as “Virus Containing Compartments (VCC)” [4]. This feature became very prominent if U1 cells are stimulated with PMA together with IFN-γ [15] or other stimuli (as reviewed in [6]) thus indicating that virion accumulation in VCC is a highly regulated process as also observed in primary MDM. Indeed, we have demonstrated, both in PMA-differentiated U1 cells and in primary MDM, that shortterm (up to 30 min) cell stimulation with extracellular ATP

U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible. . .

19

(eATP) induces the rapid discharge of VCC-accumulated virions without causing cell death via interaction with its purinergic receptor P2X7 [16]. Furthermore, Imipramine (an antidepressant agent known to interfere with microvesicle generation from the plasma membrane) blocked eATP-induced release of virions both in primary MDM and in PMA-differentiated U1 cells [16] suggesting that microvesicles could be a source of VCC-associated virions. A recent study [17] has demonstrated that both U1 and ACH-2 cells line are characterized by multiple integration sites indicative of multiple cycles of reinfection without, however, changing the overall phenotype and responsiveness of these cell lines to exogenous stimuli. Other inducers and inhibitors of HIV reactivation in the U1 cell lines are shown in Table 1. 1.2 OM-10.1 Cell Line

The chronically HIV-1 infected OM-10.1 cell line, harboring a single proviral copy per cell, was generated by limiting dilution cloning of OM cells surviving the acute infection of their parental HL-60 promyelocytic cell line that was cocultured with γ-irradiated A3.01 T cell line (a derivative of the CEM cell line) infected with HIV-1LAI/IIIB [46, 47]. As other chronically infected cells lines, its basal low-levels of HIV-1 expression could be significantly boosted by short-term stimulation with either PMA or TNF-α [48]. Later studies by Butera and Folks showed that the signaling receptor involved in TNF-mediated induction of virus expression was p55, however with and indirect “ligand passing” role also of the p75 receptor [49]. As observed in U1 and ACH-2 cells [12], endogenous TNF-α acted in an autocrine fashion in OM-10.1 cells, whereas soluble TNF receptors blocked its action [50]. A unique feature of this cell line is its maintenance, while chronically infected, of a significant proportion of cells expressing CD4 at the cell surface. Virus production stimulated by these agents coincides with a profound, almost complete, downregulation of CD4 from the cell surface and intracellular accumulation of gp160/120 Env in the absence of cytopathic effects; CD4 expression reemerged after cessation of the effect of the inductive stimuli [47]. This phenotype was also associated with accumulation of unintegrated HIV DNA in this and other (J1, ACH-2) cell lines that was prevented by either 30 -azido-30 -deoxythymidine (AZT) or soluble CD4 indicating the existence of a continuous, noncytopathic process of reinfection [46] that, nonetheless, does not affect the overall functional features of this cell line. By exploiting OM-10.1 cells along with other chronically infected T cell lines (ACH-2 and 8E5), Folks and colleagues described that certain flavonoids could inhibit TNF-α dependent reactivation of HIV-1 expression [51], an effect associated with a parallel decreased accumulation of viral transcripts, but not with the

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Guido Poli

Table 1 Inducers and inhibitors of HIV-1 reactivation in U1 cells Inducer

Inhibitor a

Mechanism

References

PMA

PARP inhibitors

Transcriptional, NF-kB independent

[18]

PMA

α-Melanocytestimulating hormone peptides

Transcriptional, NF-kB dependent

[19]

PMA

Transcriptional, Tat-associated TAK [20] activation

PMA

Cyclopentenone Prostaglandins (cyPG)

PPARγa-dependent cyPG

[21]

PMA

BRD4-selective small molecule modulator (ZL0580)

Epigenetic repression

[22]

PMA

miR-29a downregulation

[23] a

TNF-α

Transcriptional, p38 MAPK /C/ EBPβ

[24]

TNF-α

Statins

Inhibition of protein prenylation

[25]

TNF-α

Ku

Transcriptional repression

[26]

TNF-α

Hydroxyurea

NF-kB inhibition

[27]

TNF-α

Benzothiophene derivatives

Transcriptional, NF-kBindependent

[28]

TNF-α, IL-1β, Cholera toxin

Transcriptional, cAMP/AP-1, CREB/ATF

[29]

TNF-α, cell cycle synchronization

Not explored

[30]

Transcriptional, cAMP/SP-1

[31]

Transcriptional inhibition by C-terminally truncated STAT5/ NF-kB1(p50)

[32]

SP-1, SP-3

[27]

Forskolin GM-CSF

Mithramycin A

a

IL-6 + hydroxyurea Prostratin (PKCa activator)

Ricin A chain containing Elimination of infected cells nanocapsules

[33]

Estrogens, progesterone

Not explored

[34]

Epigenetic

[35]

Nitric oxide (NO)

Not explored

[36]

Mutant Cas9transcriptional activator and guide RNAs

Transcriptional

[37]

Soluble Nef protein

Not explored

[38]

Gardnerella vaginalis HDAC inhibitors, BSO

a

(continued)

U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible. . .

21

Table 1 (continued) Inducer

Mechanism

References

VpR

Transcriptional, multiple pathways

[39]

HIV-1, HIV-2 superinfection

Transcriptional, multiple pathways

[40]

Apoptosis-dependent HIV reactivation

[41]

Polo-like kinase/ bromodomain inhibitors

Epigenetic

[42]

Mesenchymal stem cells

Noncanonical PI3K-NF-kB pathway

[43]

Sodium butyrate

Transcriptional, NF-kBindependent

[44]

Cytotoxic drugs

Genome-wide CRISPR screening approach

Inhibitor

Z-VAD-FMK

TSC1 and DEPDC5 AKT-mTOR–signaling pathway (mTORC1ainhibitors)

[45]

PARP Poly[ADP-ribose] polymerase 1, PPARγ peroxisome proliferator–activated receptor γ, MAPK mitogen-activated protein kinase, GM-CSF granulocyte–macrophage colony stimulating factor, PKC protein kinase C, BSO buthionine sulfoximine, mTORC1 mammalian target of rapamycin complex 1 a

inhibition of NF-kB activation [51]. Later, independent studies demonstrated the capacity of some flavonoids to interfere with Tat-dependent transcriptional elongation, as reviewed in [52]. Other applications of the OM-10.1 cell lines are summarized in Table 2.

2

Materials

2.1 Complete Medium

2.2 Well-Established Latency-Reversing Agents Active on U1 and OM-10.1 Cells

U1 and OM-10.1 cells are typically maintained in RPMI 1640 medium supplemented with 10% FBS or FCS, glutamine (2 mM/ L), and nonessential amino acids (ca. 1 g/L). 1. Phorbol esters (PMA, PDBU). In the case of PMA, its stock should be dissolved in either DMSO or ethanol and stored at
80  C or
20  C. (a) Dissolve in complete medium in order to obtain a 100-fold higher concentration vs. the final concentration in cell culture, ranging from 10
7 to 10
9 M. For example, dilute 10 μl of concentrated solution into the cell suspension in a total volume of 990 μl. NB: PMA is a lipophilic compound that enters the cells and cannot be removed by cell centrifugation.

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Guido Poli

Table 2 Inducers and inhibitors of HIV-1 reactivation in OM-10.1 cells Inducer

Inhibitor

Mechanism

References

PMA

BRD4-selective small molecule modulator (ZL0580)

Disruption of Tat/CDK9 interaction

[22]

PMA, calcium ionophore (A23187)

Calpain inhibitor

NF-kB dependent viral transcription

[53]

PMA, TNF-α

Acridone derivativesa

PKC (NF-kB) inhibition

[54]

Galectin-3 expression, potential interaction with Tata

[55]

PMA, TNF-α PMA, TNF-α

PD121871, PD144795 (benzothiophene derivatives)

Tat and NF-kB independent viral transcription

[28]

TNF-α

MazFb

Decreased viral RNAs, apoptosis induction

[56]

TNF-α

CDK2-RNA interference Apoptosis induction

[57]

TNF-α

Aurothioglucose

NF-kB dependent viral transcription

[58]

TNF-α

JTK-101 (naphthalene derivative)

Interference with Tat-dependent transcription

[59]

TNF-α

Topotecan (topoisomerase inhibitor)

Not explored

[60]

TNF-α

Transcriptional inhibition in vitro and in [61] HM-12 and HM-13 SCID mice engrafted with OM-10.1 (6-desfluoroquinolone cells derivatives)

TNF-α

Noraristeromycin

Inhibition of NF-kB dependent transcription

[62]

G9a (H3K9 TNF-α, Tat, methyltransferase) BIX01294 (methyltransferase inhibitor)

Transcriptional repression

[63]

Sodium butyrate

Upregulation of NCoA3 (nuclear receptor coactivator 3), downregulation of IRF8 (interferon regulatory factor 8)a

[64]

Serum Vpra

[65]

Cross-linking of adhesion molecules

Autocrine TNF-α loop

[66]

Cryptococcus neoformans and Candida albicans

Autocrine TNF-α loop

[67]

(continued)

U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible. . .

23

Table 2 (continued) Inducer

Inhibitor

Ceramide

Mechanism

References

Increased transcription

[68] [69]

PTK (protein tyrosine kinase)

Genistein

Cell cycle arrest (G2), inhibition of topoisomerase II and of Phosphatidyl-inositol turnover

Heat shock

Staurosporine

Activation of PKC and other mediators [70]

Morphine withdrawal

Inhibition of multiple restriction factors [71]

a

Also effective in U1 and/or ACH-2 cells MazF is an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli

b

(b) Soluble phorbol esters such as PDBU (Phorbol 12,13dibutyrate) can be used in substitution of PMA at the same final concentrations for experiments aiming at investigating the kinetics of cell stimulation. 2. TNF-α (or TNF-β) and other cytokines (see main text and Table 1). Cytokines should be thawed from aliquots stored at
80  C or
20  C in the same day of the planned experiment. (a) The cytokine should be diluted in complete medium in order to obtain a 100-fold higher concentration vs. the final concentration in cell culture, ranging from 0.1 to 1 ng/ml.

3

Methods 1. For experiments aiming at investigating latency reversal by detection of RT activity or p24 Gag antigen in culture supernatants, cells should be seeded at 1–2  105 cells/ml in complete medium in the presence or absence of known stimuli (see below) and allowed to proliferate for 2–4 days without need of replacing the tissue culture medium. 2. For shorter experiments, for example for studying HIV RNAs, cell concentrations of 0.5–1  106 cells are recommended. 3. Latency reversal can be typically studied in flat-bottom 96-well plates allowing multiple replicas of the identical condition; it is suggested to design experiments with 3 or more replicas per single condition. 4. At the desired times after stimulation, 10–20 μl aliquots of cell culture supernatants can be harvested without need of replacement. At the end of the experiment, cells can be harvested from cell suspension, centrifuged, resuspended at the desired cell

24

Guido Poli

concentration and volume in order to investigate cellassociated viral and cellular proteins or RNA, as described. Peak levels of viral RNA after cell stimulation are typically observed between 6 and 12 h after cell stimulation; viral proteins accumulate between 24 and 48 h poststimulation. References 1. Folks TM, Justement J, Kinter A, Dinarello CA, Fauci AS (1987) Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 238(4828): 800–802. https://doi.org/10.1126/science. 3313729 2. Emiliani S, Fischle W, Ott M, Van Lint C, Amella CA, Verdin E (1998) Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line. J Virol 72(2):1666–1670 3. Cannon P, Kim SH, Ulich C, Kim S (1994) Analysis of tat function in human immunodeficiency virus type 1-infected low-level-expression cell lines U1 and ACH-2. J Virol 68(3): 1993–1997. https://doi.org/10.1128/JVI. 68.3.1993-1997.1994 4. Folks TM, Justement J, Kinter A, Schnittman S, Orenstein J, Poli G, Fauci AS (1988) Characterization of a promonocyte clone chronically infected with HIV and inducible by 13-phorbol-12-myristate acetate. J Immunol 140(4):1117–1122 5. Griffin GE, Leung K, Folks TM, Kunkel S, Nabel GJ (1989) Activation of HIV gene expression during monocyte differentiation by induction of NF-kappa B. Nature 339(6219): 70–73. https://doi.org/10.1038/339070a0 6. Cassol E, Alfano M, Biswas P, Poli G (2006) Monocyte-derived macrophages and myeloid cell lines as targets of HIV-1 replication and persistence. J Leukoc Biol 80(5):1018–1030. https://doi.org/10.1189/jlb.0306150 7. Poli G, Bressler P, Kinter A, Duh E, Timmer WC, Rabson A, Justement JS, Stanley S, Fauci AS (1990) Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor alpha by transcriptional and post-transcriptional mechanisms. J Exp Med 172(1):151–158. https://doi.org/10. 1084/jem.172.1.151 8. Rizzi C, Crippa MP, Jeeninga RE, Berkhout B, Blasi F, Poli G, Alfano M (2006) Pertussis toxin B-oligomer suppresses IL-6 induced HIV-1 and chemokine expression in chronically infected U1 cells via inhibition of activator protein 1. J Immunol 176(2):999–1006.

https://doi.org/10.4049/jimmunol.176. 2.999 9. Poli G, Orenstein JM, Kinter A, Folks TM, Fauci AS (1989) Interferon-alpha but not AZT suppresses HIV expression in chronically infected cell lines. Science 244(4904): 575–577. https://doi.org/10.1126/science. 2470148 10. Hotter D, Sauter D, Kirchhoff F (2013) Emerging role of the host restriction factor tetherin in viral immune sensing. J Mol Biol 425(24):4956–4964. https://doi.org/10. 1016/j.jmb.2013.09.029 11. Poli G, Kinter AL, Justement JS, Bressler P, Kehrl JH, Fauci AS (1992) Retinoic acid mimics transforming growth factor beta in the regulation of human immunodeficiency virus expression in monocytic cells. Proc Natl Acad Sci U S A 89(7):2689–2693. https://doi.org/ 10.1073/pnas.89.7.2689 12. Poli G, Kinter A, Justement JS, Kehrl JH, Bressler P, Stanley S, Fauci AS (1990) Tumor necrosis factor alpha functions in an autocrine manner in the induction of human immunodeficiency virus expression. Proc Natl Acad Sci U S A 87(2):782–785. https://doi.org/10. 1073/pnas.87.2.782 13. Goletti D, Kinter AL, Hardy EC, Poli G, Fauci AS (1996) Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells. J Immunol 156(9):3501–3508 14. Goletti D, Kinter AL, Coccia EM, Battistini A, Petrosillo N, Ippolito G, Poli G (2002) Interleukin (IL)-4 inhibits phorbol-ester induced HIV-1 expression in chronically infected U1 cells independently from the autocrine effect of endogenous tumour necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist. Cytokine 17(1):28–35. https://doi.org/10.1006/ cyto.2001.0989 15. Biswas P, Poli G, Kinter AL, Justement JS, Stanley SK, Maury WJ, Bressler P, Orenstein JM, Fauci AS (1992) Interferon gamma induces the expression of human immunodeficiency virus in persistently infected promonocytic cells (U1) and redirects the production of

U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible. . . virions to intracytoplasmic vacuoles in phorbol myristate acetate-differentiated U1 cells. J Exp Med 176(3):739–750. https://doi.org/10. 1084/jem.176.3.739 16. Graziano F, Desdouits M, Garzetti L, Podini P, Alfano M, Rubartelli A, Furlan R, Benaroch P, Poli G (2015) Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages. Proc Natl Acad Sci U S A 112(25):E3265–E3273. https://doi.org/10.1073/pnas.1500656112 17. Symons J, Chopra A, Malatinkova E, De Spiegelaere W, Leary S, Cooper D, Abana CO, Rhodes A, Rezaei SD, Vandekerckhove L, Mallal S, Lewin SR, Cameron PU (2017) HIV integration sites in latently infected cell lines: evidence of ongoing replication. Retrovirology 14(1):2. https:// doi.org/10.1186/s12977-016-0325-2 18. Kameoka M, Tanaka Y, Ota K, Itaya A, Yoshihara K (1999) Poly (ADP-ribose) polymerase is involved in PMA-induced activation of HIV-1 in U1 cells by modulating the LTR function. Biochem Biophys Res Commun 262(1): 285–289. https://doi.org/10.1006/bbrc. 1999.1146 19. Barcellini W, Colombo G, La Maestra L, Clerici G, Garofalo L, Brini AT, Lipton JM, Catania A (2000) Alpha-melanocyte-stimulating hormone peptides inhibit HIV-1 expression in chronically infected promonocytic U1 cells and in acutely infected monocytes. J Leukoc Biol 68(5):693–699 20. Yang X, Gold MO, Tang DN, Lewis DE, Aguilar-Cordova E, Rice AP, Herrmann CH (1997) TAK, an HIV tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines. Proc Natl Acad Sci U S A 94(23):12331–12336 21. Hayes MM, Lane BR, King SR, Markovitz DM, Coffey MJ (2002) Peroxisome proliferator-activated receptor gamma agonists inhibit HIV-1 replication in macrophages by transcriptional and post-transcriptional effects. J Biol Chem 277(19):16913–16919. https:// doi.org/10.1074/jbc.M200875200 22. Alamer E, Zhong C, Liu Z, Niu Q, Long F, Guo L, Gelman BB, Soong L, Zhou J, Hu H (2020) Epigenetic suppression of HIV in myeloid cells by the BRD4-selective small molecule modulator ZL0580. J Virol 94(11): e01880–e01819. https://doi.org/10.1128/ JVI.01880-19 23. Patel P, Ansari MY, Bapat S, Thakar M, Gangakhedkar R, Jameel S (2014) The microRNA miR-29a is associated with human

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NF-kappaB1/p50 and C-terminally truncated STAT5 complex. J Mol Biol 410(5):933–943. https://doi.org/10.1016/j.jmb.2011.03.044 33. Wen J, Yan M, Liu Y, Li J, Xie Y, Lu Y, Kamata M, Chen IS (2016) Specific elimination of latently HIV-1 infected cells using HIV-1 protease-sensitive toxin Nanocapsules. PLoS One 11(4):e0151572. https://doi.org/ 10.1371/journal.pone.0151572 34. Simoes JA, Hashemi FB, Aroutcheva AA, Heimler I, Spear GT, Shott S, Faro S (2001) Human immunodeficiency virus type 1 stimulatory activity by Gardnerella vaginalis: relationship to biotypes and other pathogenic characteristics. J Infect Dis 184(1):22–27. https://doi.org/10.1086/321002 35. Savarino A, Mai A, Norelli S, El Daker S, Valente S, Rotili D, Altucci L, Palamara AT, Garaci E (2009) "shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence. Retrovirology 6:52 36. Mannick JB, Stamler JS, Teng E, Simpson N, Lawrence J, Jordan J, Finberg RW (1999) Nitric oxide modulates HIV-1 replication. J Acquir Immune Defic Syndr 22(1):1–9. https://doi.org/10.1097/00042560199909010-00001 37. Kim V, Mears BM, Powell BH, Witwer KW (2017) Mutant Cas9-transcriptional activator activates HIV-1 in U1 cells in the presence and absence of LTR-specific guide RNAs. Matters (Zur) 2017. https://doi.org/10.19185/ matters.201611000027 38. Fujinaga K, Zhong Q, Nakaya T, Kameoka M, Meguro T, Yamada K, Ikuta K (1995) Extracellular Nef protein regulates productive HIV-1 infection from latency. J Immunol 155(11):5289–5298 39. Varin A, Decrion AZ, Sabbah E, Quivy V, Sire J, Van Lint C, Roques BP, Aggarwal BB, Herbein G (2005) Synthetic Vpr protein activates activator protein-1, c-Jun N-terminal kinase, and NF-kappaB and stimulates HIV-1 transcription in promonocytic cells and primary macrophages. J Biol Chem 280(52): 42557–42567. https://doi.org/10.1074/jbc. M502211200 40. Wang X, Sun B, Mbondji C, Biswas S, Zhao J, Hewlett I (2017) Differences in activation of HIV-1 replication by superinfection with HIV-1 and HIV-2 in U1 cells. J Cell Physiol 232(7):1746–1753. https://doi.org/10. 1002/jcp.25614 41. Khan SZ, Hand N, Zeichner SL (2015) Apoptosis-induced activation of HIV-1 in

latently infected cell lines. Retrovirology 12: 42. https://doi.org/10.1186/s12977-0150169-1 42. Gohda J, Suzuki K, Liu K, Xie X, Takeuchi H, Inoue JI, Kawaguchi Y, Ishida T (2018) BI-2536 and BI-6727, dual polo-like kinase/ bromodomain inhibitors, effectively reactivate latent HIV-1. Sci Rep 8(1):3521. https://doi. org/10.1038/s41598-018-21942-5 43. Chandra PK, Gerlach SL, Wu C, Khurana N, Swientoniewski LT, Abdel-Mageed AB, Li J, Braun SE, Mondal D (2018) Mesenchymal stem cells are attracted to latent HIV-1infected cells and enable virus reactivation via a non-canonical PI3K-NFkappaB signaling pathway. Sci Rep 8(1):14702. https://doi. org/10.1038/s41598-018-32657-y 44. Laughlin MA, Chang GY, Oakes JW, Gonzalez-Scarano F, Pomerantz RJ (1995) Sodium butyrate stimulation of HIV-1 gene expression: a novel mechanism of induction independent of NF-kappa B. J Acquir Immune Defic Syndr Hum Retrovirol 9(4):332–339 45. Jin S, Liao Q, Chen J, Zhang L, He Q, Zhu H, Zhang X, Xu J (2018) TSC1 and DEPDC5 regulate HIV-1 latency through the mTOR signaling pathway. Emerg Microbes Infect 7(1):138. https://doi.org/10.1038/s41426018-0139-5 46. Besansky NJ, Butera ST, Sinha S, Folks TM (1991) Unintegrated human immunodeficiency virus type 1 DNA in chronically infected cell lines is not correlated with surface CD4 expression. J Virol 65(5):2695–2698 47. Butera ST, Perez VL, Wu BY, Nabel GJ, Folks TM (1991) Oscillation of the human immunodeficiency virus surface receptor is regulated by the state of viral activation in a CD4+ cell model of chronic infection. J Virol 65(9): 4645–4653. https://doi.org/10.1128/JVI. 65.9.4645-4653.1991 48. Butera ST, Roberts BD, Lam L, Hodge T, Folks TM (1994) Human immunodeficiency virus type 1 RNA expression by four chronically infected cell lines indicates multiple mechanisms of latency. J Virol 68(4): 2726–2730. https://doi.org/10.1128/JVI. 68.4.2726-2730.1994 49. Butera ST, Roberts BD, Folks TM (1996) Ligand passing by the p75 tumour necrosis factor receptor enhances HIV-1 activation. Cytokine 8(10):745–750. https://doi.org/ 10.1006/cyto.1996.0099 50. Butera ST, Roberts BD, Folks TM (1993) Regulation of HIV-1 expression by cytokine networks in a CD4+ model of chronic infection. J Immunol 150(2):625–634

U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible. . . 51. Critchfield JW, Butera ST, Folks TM (1996) Inhibition of HIV activation in latently infected cells by flavonoid compounds. AIDS Res Hum Retrovir 12(1):39–46. https://doi.org/10. 1089/aid.1996.12.39 52. Wang Y, Liu XY, De Clercq E (2009) Role of the HIV-1 positive elongation factor P-TEFb and inhibitors thereof. Mini Rev Med Chem 9(3):379–385. https://doi.org/10.2174/ 1389557510909030379 53. Teranishi F, Liu ZQ, Kunimatsu M, Imai K, Takeyama H, Manabe T, Sasaki M, Okamoto T (2003) Calpain is involved in the HIV replication from the latently infected OM10.1 cells. Biochem Biophys Res Commun 303(3): 940–946. https://doi.org/10.1016/s0006291x(03)00447-9 54. Fujiwara M, Okamoto M, Okamoto M, Watanabe M, Machida H, Shigeta S, Konno K, Yokota T, Baba M (1999) Acridone derivatives are selective inhibitors of HIV-1 replication in chronically infected cells. Antivir Res 43(3):189–199. https://doi.org/10. 1016/s0166-3542(99)00045-5 55. Okamoto M, Hidaka A, Toyama M, Baba M (2019) Galectin-3 is involved in HIV-1 expression through NF-kappaB activation and associated with tat in latently infected cells. Virus Res 260:86–93. https://doi.org/10.1016/j. virusres.2018.11.012 56. Okamoto M, Chono H, Hidaka A, Toyama M, Mineno J, Baba M (2020) Induction of E. coliderived endonuclease MazF suppresses HIV-1 production and causes apoptosis in latently infected cells. Biochem Biophys Res Commun 530(3):597–602. https://doi.org/10.1016/j. bbrc.2020.07.103 57. Ammosova T, Berro R, Kashanchi F, Nekhai S (2005) RNA interference directed to CDK2 inhibits HIV-1 transcription. Virology 341(2):171–178. https://doi.org/10.1016/j. virol.2005.06.041 58. Traber KE, Okamoto H, Kurono C, Baba M, Saliou C, Soji T, Packer L, Okamoto T (1999) Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells. Int Immunol 11(2): 143–150. https://doi.org/10.1093/intimm/ 11.2.143 59. Wang X, Yamataka K, Okamoto M, Ikeda S, Baba M (2007) Potent and selective inhibition of tat-dependent HIV-1 replication in chronically infected cells by a novel naphthalene derivative JTK-101. Antivir Chem Chemother 18(4):201–211. https://doi.org/10.1177/ 095632020701800404

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Part II In Vitro and Ex Vivo Primary Cell Models of Minimal but Inducible Proviral Expression

Chapter 3 An In Vitro System to Model the Establishment and Reactivation of HIV-1 Latency in Primary Human CD4+ T Cells Rui Li and Fabio Romerio Abstract HIV-1 establishes latency primarily by infecting activated CD4+ T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. Here we describe both the original and a simplified version of HIV-1 latency models that mimics this process using replication competent viruses. Our model allows generation of large numbers of latently infected CD4+ T cell to dissect molecular mechanisms of HIV latency and reactivation. Key words HIV-1, Latency, Monocyte-derived dendritic cells, CD4+ T cells

1

Introduction HIV-1 establishes a status of nonproductive infection known as viral latency [1]. Harper et al. reported that cells harboring the provirus in clinical samples were found at frequency considerably higher than cells expressing viral RNA, thus providing the first indirect evidence for the existence of latently infected cells in HIV-1 patients [2]. Formal demonstration came from Siliciano and colleagues [3], who detected integrated, latent HIV-1 genome in resting peripheral blood CD4+ T cells of HIV-1 patients. Latently infected cells persist in patients undergoing antiretroviral therapy and with undetectable viremia, and HIV-1 can be rescued in vitro from the cells of these patients [4–6]. The rate of decay for the latently infected pool has been calculated at ~44 months, sufficiently slow to guarantee lifelong persistence and to preclude HIV-1 eradication with HAART [7]. Moreover, as a dormant provirus, HIV-1 is invisible to cellular and humoral immune responses and resistant to drugs interfering with its life cycle [8].

Guido Poli et al. (eds.), HIV Reservoirs: Methods and Protocols, Methods in Molecular Biology, vol. 2407, https://doi.org/10.1007/978-1-0716-1871-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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HIV-1 establishes latency by exploiting the cellular events of CD4+ T cell blast transformation, clonal expansion, and clonal contraction that lead to the formation of immunological memory [9]. During the contraction phase at the end of the immune response, the vast majority of activated CD4+ T cells die in response to intrinsic mechanisms triggered by antigen (Ag) withdrawal and consequent decline of growth factors and Bcl-2, as well as extrinsic mechanisms involving Fas (CD95)-Fas ligand (CD178) interaction and caspase 3 activation [10]. Production of interleukin (IL)-7 during the contraction phase promotes the establishment and maintenance of immunological memory through the up-regulation of Bcl-2 expression, which counteracts the intrinsic pathway of activated CD4+ T cell apoptosis [11–13]. HIV-1 typically infects cells during blast transformation and clonal expansion [14, 15]. Although the majority of HIV-1-infected lymphoblast CD4+ T cells die as a consequence of apoptosis or cytopathic effects of HIV-1, some infected cells survive clonal contraction and return to the resting state as memory cells bearing an integrated HIV-1 genome. In their resting state, HIV-1-infected cells do not actively replicate the virus, which remains dormant for extended periods of time [16, 17]. Viral production resumes following Ag-driven proliferation and cytokine stimulation. Relying the study of HIV-1 latency on clinical samples and chronically infected cell lines presents severe limitations such as the very low frequency of latently infected cells in the former and the lack of physiological relevance of the latter [8]. In addition, systems based on the use of clinical samples allow us to study HIV-1 reactivation from latency, but not how latency is established. Here, we describe a method to generate resting nonproductively infected CD4+ T cells carrying a replication-competent HIV-1 genome following Ag-driven proliferation in vitro [18–20]. This system may help shed light onto the mechanisms of HIV-1 latency, and lead to identify new therapeutic strategies.

2

Materials

2.1 Cell Culture Media and Reagents

1. R10 medium: RPMI 1640 supplemented with 1% penicillin/ streptomycin/L-glutamine, and 10% human serum AB (HSAB). Pass through a 0.22-μm sterile filter prior to use. 2. MDDC medium: RPMI 1640 supplemented with 1% penicillin/streptomycin/L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 50 μM 2-mercaptoethanol (ThermoFisher), 50 μg/ml gentamicin, 10% HSAB. Pass through a 0.22-μm sterile filter prior to use. 3. Staphylococcal Enterotoxin B (SEB). 4. Recombinant human IL-2 (rhIL-2).

An In Vitro System to Model the Establishment and Reactivation of HIV-1. . .

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5. rhIL-7, rhGM-CSF, and rhIL-4. 6. Human T-Activator CD3/CD28 Dynabeads. 2.2 Cell Isolation Kits, Supplies, and Buffers

1. Monocyte, Naı¨ve CD4+ T cell, Memory CD4+ T cell, and Total CD4+ T cell Isolation Kits (Miltenyi Biotec). 2. LS or MS magnetic separation columns (Miltenyi Biotec). 3. MACS Separator magnets and stand (Miltenyi Biotec). 4. Running buffer: 2 mM EDTA, 0.5% bovine serum albumin (BSA) in 1 PBS. Pass through a 0.22-μm sterile filter prior to use. 5. Cell Dissociation Buffer (CDB): 2 mM EDTA and 2% FBS in 1 PBS. Pass through a 0.22-μm sterile filter prior to use.

2.3

Flow Cytometry

1. Cell Purity antibody panel: anti-CD3, CD4, CD8, CD14, CD16, CD19, CD56, HLA-DR, TCRγδ. 2. Fresh and Activated CD4+ T cell antibody panel: anti-CD25, CD45R0, CD62L, CD69, HLA-DR. 3. Monocyte and MDDC antibody panel: anti-CD14, CD80, CD86, HLA-DR. 4. Rested CD4+ T cell antibody panel: anti-CD25, CD62L, HLA-DR, and CCR7. 5. Cell Cycle panel: anti-Ki67 and 7-amino-actinomycin D. 6. HIV-1 infection (clone KC57).

antibody:

anti-HIV-1

p24

antigen

7. Cytofix/Cytoperm kit. 8. Wash buffer: 2% fetal bovine serum in 1 PBS. Pass through a 0.22-μm sterile filter prior to use. 9. Fixation buffer: 2% paraformaldehyde (PFA) in 1 PBS. 10. Permeabilization Buffer: 0.05% saponin in wash buffer. 2.4

HIV-1 p24 ELISA

1. HIV-1 p24 ELISA Kit. 2. ELISA lysis buffer: 0.5% Triton X-100, 0.002% sodium azide in 1 PBS.

3

Methods The model of HIV-1 latency we have developed recapitulates in vitro the events of blast transformation, clonal expansion, and clonal contraction leading to the generation of resting, memory cells [18–20]. It consists of four steps (Fig. 1a): (1) generation of immature dendritic cells from primary monocytes (iMDDC), (2) activation of naı¨ve CD4+ T cells with MDDC and antigen,

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Fig. 1 Generation of latently infected CD4+ T cells in vitro. (a) Experimental procedure for the generation of resting nonproductively infected CD4+ T cells. (b) Flow cytometry analysis of naı¨ve CD4+ T cells enriched by negative selection from PBMCs. (c) Flow cytometry analysis of CD4+ T cells after activation with MDDC. All plots (except for FSC-H vs. SSC-H) are gated in the live cell population as determined by forward and side light scatter profile and exclusion of 7AAD. The days of analysis reported in both panels match the timescale reported in panel a. Results are representative of more than 20 different donors

(3) HIV-1 infection and expansion of activated CD4+ T cells, and (4) generation of resting, nonproductively infected cells. A fifth step involves the reactivation (secondary stimulation) of resting, nonproductively infected cells with either MDDC plus SEB or immobilized anti-CD3/CD28 Abs leading to rescue of latent provirus. 3.1 Day 0: Generation of Immature MonocyteDerived Dendritic Cells (iMDDC)

1. Isolate peripheral blood mononuclear cells (PBMCs) from the buffy coat of healthy donors by Ficoll density gradient centrifugation, and enrich CD14+ monocytes from PBMCs by negative selection using Monocyte Isolation Kit per manufacturer’s protocols (Fig. 1a). 2. Analyze cells by flow cytometry with the Cell Purity antibody panel (see Note 1). Transfer 2.5  105 freshly isolated cells to a microcentrifuge tube using as many tubes needed to cover the entire antibody panel plus isotype control antibodies. Wash the

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cells with wash buffer. Resuspend the cells in 250 μl of wash buffer, add appropriate volume of each antibody, and incubate on ice for 30 min. Wash the cells twice with wash buffer. Fix with fixation buffer and analyze by flow cytometry. 3. Analyze cells by flow cytometry with the Monocyte and MDDC antibody panel (see Note 2). 4. Resuspend isolated CD14+ monocytes at 1  106/ml in MDDC medium without serum and cytokines, plate in 6-well plates at 3  106 cells/well, and incubate at 37  C for 2 h. 5. Remove culture supernatant with nonadherent cells, and wash adherent cells extensively with MDDC medium to remove all residual nonadherent cells. 6. Culture adherent cells in complete MDDC medium supplemented with 50 ng/ml rhGM-CSF and 50 ng/ml rhIL-4 for 5 days at 37  C with a 50% medium change at day 3. 7. Recover detached and loosely attached MDDC by pipetting, and transfer to a centrifuge tube. 8. To recover still adherent iMDDC, add 1–2 ml of ice-cold CDB to each well and incubate on ice for 15–20 min. As adherent cells round up and become loosely attached, recover them by vigorous pipetting, and mix with the cells from step 6 above (see Note 3). 9. Centrifuge and resuspend the cells in complete MDDC medium. 10. Analyze iMDDC by flow cytometry with the Monocyte and MDDC antibody panel (see Note 2). 3.2 Day 5: Activation of Naı¨ve CD4+ T Cells

1. Enrich naive CD4+ T cells from autologous PBMCs by negative selection using the Naı¨ve CD4+ T cell Isolation Kit (Fig. 1a; see Note 4). 2. Analyze cells by flow cytometry using the Cell Purity antibody panel following the same staining protocol described in the previous section (see Note 5). 3. Analyze the cells with the Fresh and Activated CD4+ T cell antibody panel and the Cell Cycle panel (Fig. 1b). For the latter panel, fix and permeabilize the cells using the Cytofix/Cytoperm Kit following the manufacturer’s instructions (see Note 5). 4. Resuspend naı¨ve CD4+ T cells at 1  106/ml, mix with iMDDC (from step 9 in previous section) at a 10:1 (naı¨ve– MDDC) ratio, and culture in complete MDDC medium supplemented with 250 ng/ml SEB and 25 U/ml rhIL-2 for 4 days (see Notes 6–8).

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3.3 Day 9: HIV-1 Infection and Expansion of Activated CD4+ T Cells

1. Recover activated CD4+ T cells by pipetting and wash with R10 medium without HSAB. 2. Analyze cells using the Fresh and Activated CD4+ T cell antibody panel and the Cell Cycle panel following the same procedures described above (Fig. 1c; see Note 9). 3. Wash and resuspend the activated CD4+ T cells in R10 medium without HSAB at 3  106 cells/ml. 4. Infect the activated CD4+ T cells with replication competent HIV-1 virus at a multiplicity of infection (MOI) of 0.05 for 2 h at 37  C (see Note 10). 5. Wash cells 3–5 times with complete R10 medium to remove cell-free virus. 6. Resuspend the cells at 0.5  106 cells/ml in complete R10 medium supplemented with 25 U/ml rhIL-2. 7. Every 3 days and over a period of 15 days, dilute cells to 1  106 cells/ml by adding fresh complete R10 medium supplemented with 25 U/ml IL-2 (see Note 11). 8. During the expansion phase, monitor viral replication by HIV-1 p24 ELISA in the culture supernatants and in cell lysates (Fig. 2a). For the latter, transfer 1  105 live cells in a microcentrifuge tube, wash twice with 1 PBS, lyse the cell pellet with 200 μl of ELISA lysis buffer. 9. During the expansion phase, also monitor viral replication by HIV-1 p24 intracellular flow cytometry (Fig. 2b). Aliquot 1  106 cells into two 1.5 ml microcentrifuge tubes, wash with flow cytometry wash buffer, resuspend in 200 μl of permeabilization buffer, and incubate at 4  C for 10 min. Add 1 μl of anti-p24 KC57 or IgG isotype control antibody to each tube. Incubate at 4  C for 30 min. Wash twice with flow cytometry wash buffer, fix with 0.5 ml of fixation buffer. Analyze by flow cytometry (see Note 12).

3.4 Day 24: Isolation of Memory CD4+ T Cells and Generation of Resting Cells

1. Enrich CD4+ T cells by negative selection using the Memory CD4+ T cell Isolation Kit. 2. Analyze the cells by flow cytometry using the Fresh and Activated CD4+ T cell antibody panel and the Cell Cycle panel as described above (Fig. 3a). 3. Culture enriched CD4+ T cells in complete R10 medium supplemented with 1 ng/ml rhIL-7 for ~4 weeks performing a 50% medium change every 2 days. 4. Analyze the cells by flow cytometry using the Rested CD4+ T cell antibody panel and the Cell Diploidy panel as described above (Fig. 3b; see Note 13).

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Fig. 2 HIV-1 replication during the phase of cell expansion. (a) Intracellular and extracellular HIV-1 p24gag levels in whole-cell extracts (closed circles) and culture supernatants (closed squares), respectively. Cell viability (percentage of live cells) determined by trypan blue exclusion is also shown in this panel (open diamonds: uninfected cultures; closed diamonds: infected cultures). (b) Intracellular flow cytometry analysis of HIV-1 p24gag antigen in infected and uninfected cultures. All flow cytometry plots are gated in the live cell population as determined by forward and side light scatter profile and exclusion of 7AAD. The days of analysis reported in both panels correspond to the timescale reported in Fig. 1a. Results are representative of 10 different donors

5. Monitor HIV-1 p24 levels both by and ELISA (Fig. 4a) and intracellular flow cytometry staining (Fig. 4c) as described above (see Note 14). 3.5 Day 53: Reactivation of HIV-1 Replication from Resting Latently Infected CD4+ T Cells

1. To reactivate HIV-1 expression, restimulate rested CD4+ T cells with SEB-loaded autologous iMDDC as described above (see Note 15). 2. Monitor HIV-1 p24 levels both by intracellular flow cytometry staining (Fig. 5b) and ELISA (Fig. 5c) as described above (see Note 16). 3. Cell reactivation can be monitored by flow cytometry using the Fresh and Activated CD4+ T cell antibody panel as described above.

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Fig. 3 Enrichment of activated (memory-phenotype) CD4+ T cells and generation of resting cells. (a) Flow cytometry analysis of activated (memory-phenotype) CD4+ T cells enriched by negative selection from CD4+ T cells and MDDC cultures. (b) Flow cytometry analysis of rested CD4+ T cells. All plots (except for FSC-H vs. SSC-H plots) are gated in the live cell population as determined by forward and side light scatter profile and exclusion of 7AAD. The days of analysis reported in both panels correspond to the timescale reported in Fig. 1a. Results are representative of more than 10 different donors

4

Notes 1. Typically, monocyte purity is 99% following cell adhesion to plastic and extensive washing that eliminate lymphocyte contaminants. 2. Freshly isolated CD14+ monocytes express HLA-DR, but very low levels of the costimulatory molecules, CD80 (B7.1) and CD86 (B7.2). Expression of costimulatory receptors is markedly upregulated on MDDC. 3. During differentiation of monocytes into dendritic cells, some cells detach from the plastic support or remain loosely attached. These can be easily harvested by pipetting. Firmly attached cells can be recovered after incubation in CDB and vigorous pipetting. 4. As an alternative to naı¨ve CD4+ T cells, total CD4+ T cells can also be used. The advantage of using total CD4+ T cells is that a significantly greater number of cells can be activated from the same number of starting PBMC. Total CD4+ T cells can be enriched from PBMC by negative selection using the Total CD4+ T cell Isolation Kit.

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39

Fig. 4 Suppression of HIV-1 replication during the resting phase. (a) Intracellular and extracellular HIV-1 p24gag levels in whole-cell extracts (closed circles) and culture supernatants (closed squares), respectively. The closed circle and closed square at day 24 refer to the intracellular and extracellular p24gag levels measured immediately prior to enrichment of activated (memory-phenotype) CD4+ T cells (see also Fig. 3a). (b) Effect of cycloheximide (CHX) and azidothymidine (AZT) on the intracellular levels of p24gag antigen during the resting phase. Closed triangles: CHX-treated cultures; open squares: AZT-treated cultures; closed circles: untreated cultures. (c) Intracellular flow cytometry analysis of HIV-1 p24gag antigen in infected and uninfected cultures. All flow cytometry plots are gated in the live cell population as determined by forward and side light scatter profile and exclusion of 7AAD. The days of analysis reported in all panels correspond to the timescale reported in Fig. 1a. Results are representative of 5–10 different donors

5. Purity of freshly isolated CD4+ T cells should be >98% as determined by flow cytometry. In addition, freshly isolated CD4+ T cells lack activation markers (CD25, CD69 and HLA-DR), and are not in cell cycle (lack of Ki67 expression, and 2N ploidy). Expression of CD45R0 is dependent on the cell population isolated (see Note 4 above): while naı¨ve cells are CD45R0-negative (CD45RA-positive), total CD4+ T cells contain both CD45R0-negative and positive populations, due to the presence of both naı¨ve and memory CD4+ T cells. 6. When using iMDDC to activate CD4+ T cells, it is important to utilize cells obtained from the same donor to avoid the occurrence of mixed lymphocyte reaction (MLR).

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Fig. 5 Analysis of HIV-1 replication during secondary stimulation. (a) Intracellular flow cytometry analysis of HIV-1 p24gag antigens at the end of the expansion and resting phases (upper and lower plots, respectively). (b) Intracellular flow cytometry analysis of HIV-1 p24gag antigen during secondary stimulation with antigen-loaded MDDC in the absence and in the presence of 1 μM AZT (plots in the upper and lower row, respectively). (c) HIV-1 p24gag levels in culture supernatants during secondary stimulation with antigen-loaded MDDC in the absence or in the presence of 1 μM AZT (closed circles and squares, respectively). All flow cytometry plots are gated in the live cell population as determined by forward and side light scatter profile and exclusion of 7AAD. The days of analysis reported in all panels correspond to the timescale reported in Fig. 1a

7. Maturation of MDDC occurs upon coculture with CD4+ T cells. 8. Human T-Activator CD3/CD28 Dynabeads can be used in place of antigen-loaded MDDC to activate resting cells prior to initial HIV-1 infection. The use of MDDC represents a more physiologically relevant approach involving the entire array of stimuli delivered by antigen-presenting cells, both via direct cell–cell interaction and via cytokine production. However, it allows activation of only a subset of cells that are able to recognize the antigen presented by the MDDC. Even superantigens such as SEB activate only ~20% of the CD4+ T cells. The use of anti-CD3/CD28-coated magnetic beads does not

An In Vitro System to Model the Establishment and Reactivation of HIV-1. . .

41

offer the array of signals delivered by antigen presenting cells, but it offers the advantage of activating nearly all CD4+ T cells. For CD4+ T cell activation using anti-CD3/CD28 Dynabeads, resuspend freshly isolated cells at 0.5  106 cells/ml in complete R10 medium containing 1 bead/cell plus 50 U/ml IL-2. After 3 days, harvest the cells, dissociate bead-cell interactions by pipetting, transfer the suspension to a sterile tube, and apply the tube to a magnetic field for 2–3 min. When the magnetic beads have migrated to the tube wall, transfer the cell suspension to a clean tube, and repeat the procedure to eliminate residual beads. Finally, count the cells and proceed with HIV-1 infection and cell culture as described above. 9. Activated cells present larger size and granularity as assessed by forward and side scatter light in flow cytometry. In addition, they show expression of activation markers (CD25, CD69 and HLA-DR), and they appear in cell cycle (expression of Ki67 and 2N–4N ploidy). If CD45R0-negative naı¨ve CD4+ T cells were the starting population, expression of CD45R0 will be detectable in the activated population. 10. The size of the viral inoculum ensures that as much as 20% of the cells will be HIV-1 infected by the end of the expansion phase. The final percentage of infected cells can be modified (either higher or lower) so as to suit the particular aims of the study. The final percentage of infected cells can be increased also by optimizing the efficiency of HIV-1 entry during primary infection. This can be achieved via addition of 10 μg/ml polybrene or HIV-1 Infectin™ (Virongy) during primary infection. An alternative, involves the use of spinoculation. For the latter, upon adding the virus inoculum to the activated CD4+ T cells, incubate the tube at 37  C for 30 min. Then centrifuge the cells at 1200  g for 2 h at room temperature. Remove the supernatant and wash the pellet several times with complete R10 medium. Proceed with the cell culture as described in Subheading 3.3. 11. Culture medium should not be replaced, because it contains viral particles able to infect new target cells and expand the fraction of the cell population carrying HIV-1. At the same time, cell density should be maintained between 1–2  106 cells/ml to facilitate cell expansion and to reduce apoptosis triggered by cell–cell interaction. 12. The frequency of infected cells at the end of the infection period is variable depending on the size of the initial inoculum and the use of infection enhancers (polybrene, HIV Infectin™, spinoculation, etc.). 13. During the resting phase, CD4+ T cells progressively exit the cell cycle (lack of Ki67 expression, and 2N ploidy), and return

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to quiescence (lack of CD25, CD69, and HLA-DR expression). Coexpression of high levels of CD62L and CCR7 indicates a central memory phenotype. 14. During the resting phase, detection of intracellular HIV-1 p24 antigen by flow cytometry may remain elevated due to the stability of the protein expressed during active viral replication. However, the declining levels of HIV-1 p24 levels in the culture supernatant will indicate the establishment of HIV-1 latency. The addition of the HIV-1 reverse transcriptase inhibitor, azidothymidine (AZT) or the protein synthesis inhibitor, cycloheximide (CHX) during the resting phase does not impact the decline of intracellular levels of HIV-1 p24 antigen (Fig. 4b). This indicates that intracellular p24 detected during this phase does not reflect new rounds of infection (which would be blocked by AZT) or de novo expression from existing proviruses (which would be blocked by CHX), but rather protein expressed during the activate viral replication in the expansion phase and persisting in the cytoplasm of infected cells. 15. Cell reactivation can also be performed with CD3/CD28 Dynabeads as described above. 16. Treatment with AZT during secondary stimulation prevents the accumulation of HIV-1 p24 antigen in the culture medium, indicating the cell stimulation reactivates HIV-1 from latent reservoirs, and leads to spread of the infection to new target cells (Fig. 5c).

Acknowledgments This work was supported by the National Institute of Health grants R21AI084711 and R21AI106508 (to F.R.). The authors would like to thank Zahra Gholizadeh for helpful discussion. References 1. Tyagi M, Romerio F (2011) Models of HIV-1 persistence in the CD4+ T cell compartment: past, present and future. Curr HIV Res 9 (8):579–587 2. Harper ME, Marselle LM, Gallo RC, WongStaal F (1986) Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Natl Acad Sci U S A 83(3):772–776 3. Chun TW, Finzi D, Margolick J, Chadwick K, Schwartz D, Siliciano RF (1995) In vivo fate of HIV-1-infected T cells: quantitative analysis of

the transition to stable latency. Nat Med 1 (12):1284–1290 4. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, Quinn TC, Chadwick K, Margolick J, Brookmeyer R, Gallant J, Markowitz M, Ho DD, Richman DD, Siliciano RF (1997) Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278 (5341):1295–1300 5. Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, Lloyd AL, Nowak MA, Fauci AS (1997) Presence of an inducible

An In Vitro System to Model the Establishment and Reactivation of HIV-1. . . HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci U S A 94(24):13193–13197. https://doi.org/10. 1073/pnas.94.24.13193 6. Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, Spina CA, Richman DD (1997) Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278(5341):1291–1295 7. Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, Smith K, Lisziewicz J, Lori F, Flexner C, Quinn TC, Chaisson RE, Rosenberg E, Walker B, Gange S, Gallant J, Siliciano RF (1999) Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5(5):512–517. https://doi.org/10. 1038/8394 8. Han Y, Wind-Rotolo M, Yang HC, Siliciano JD, Siliciano RF (2007) Experimental approaches to the study of HIV-1 latency. Nat Rev Microbiol 5(2):95–106. https://doi.org/ 10.1038/nrmicro1580 9. Jenkins MK, Khoruts A, Ingulli E, Mueller DL, McSorley SJ, Reinhardt RL, Itano A, Pape KA (2001) In vivo activation of antigen-specific CD4 T cells. Annu Rev Immunol 19:23–45. https://doi.org/10.1146/annurev.immunol. 19.1.23 10. Marrack P, Kappler J (2004) Control of T cell viability. Annu Rev Immunol 22:765–787. https://doi.org/10.1146/annurev.immunol. 22.012703.104554 11. Bradley LM, Haynes L, Swain SL (2005) IL-7: maintaining T-cell memory and achieving homeostasis. Trends Immunol 26 (3):172–176. https://doi.org/10.1016/j.it. 2005.01.004 12. Swain SL, Agrewala JN, Brown DM, JelleyGibbs DM, Golech S, Huston G, Jones SC, Kamperschroer C, Lee WH, McKinstry KK, Roman E, Strutt T, Weng NP (2006) CD4+ T-cell memory: generation and multi-faceted roles for CD4+ T cells in protective immunity to influenza. Immunol Rev 211:8–22. https:// doi.org/10.1111/j.0105-2896.2006. 00388.x

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13. Khaled AR, Durum SK (2003) Death and Baxes: mechanisms of lymphotrophic cytokines. Immunol Rev 193:48–57 14. Margolick JB, Volkman DJ, Folks TM, Fauci AS (1987) Amplification of HTLV-III/LAV infection by antigen-induced activation of T cells and direct suppression by virus of lymphocyte blastogenic responses. J Immunol 138 (6):1719–1723 15. Zack JA, Cann AJ, Lugo JP, Chen IS (1988) HIV-1 production from infected peripheral blood T cells after HTLV-I induced mitogenic stimulation. Science 240(4855):1026–1029 16. Hermankova M, Siliciano JD, Zhou Y, Monie D, Chadwick K, Margolick JB, Quinn TC, Siliciano RF (2003) Analysis of human immunodeficiency virus type 1 gene expression in latently infected resting CD4+ T lymphocytes in vivo. J Virol 77(13):7383–7392. https://doi.org/10.1128/jvi.77.13.73837392.2003 17. Lassen KG, Bailey JR, Siliciano RF (2004) Analysis of human immunodeficiency virus type 1 transcriptional elongation in resting CD4+ T cells in vivo. J Virol 78 (17):9105–9114. https://doi.org/10.1128/ JVI.78.17.9105-9114.2004 18. Marini A, Harper JM, Romerio F (2008) An in vitro system to model the establishment and reactivation of HIV-1 latency. J Immunol 181 (11):7713–7720 19. Iglesias-Ussel M, Marchionni L, Romerio F (2013) Isolation of microarray-quality RNA from primary human cells after intracellular immunostaining and fluorescence-activated cell sorting. J Immunol Methods 391 (1–2):22–30. https://doi.org/10.1016/j.jim. 2013.02.003 20. Iglesias-Ussel M, Vandergeeten C, Marchionni L, Chomont N, Romerio F (2013) High levels of CD2 expression identify HIV-1 latently infected resting memory CD4+ T cells in virally suppressed subjects. J Virol 87 (16):9148–9158. https://doi.org/10.1128/ JVI.01297-13

Chapter 4 The Cultured TCM Model of HIV Latency Alberto Bosque Abstract Models to study HIV latency have improved our understanding of the mechanisms involved in this process and have helped in the discovery and development of therapeutic strategies to eradicate HIV. Primary cell models are based on the in vitro generation of latently infected cells using CD4T cells isolated from blood, lymph nodes or other lymphoid organs. In this chapter, we describe the generation of HIV latently infected memory CD4T cells using blood naı¨ve CD4T cells from peripheral blood with a phenotype resembling that of central memory CD4T cells. This model can be used to investigate the mechanisms involved in latency as well to develop strategies to target it. Key words Latency, Central Memory CD4T cells, Replication-competent HIV, Reservoirs, Latencyreversal

1

Introduction To better understand factors that can influence the establishment and reactivation of HIV latency in primary CD4T cells, several groups have developed primary cell models of latency [1–6]. We first described a primary cell model based on the in vitro generation of central memory CD4 T cells using naı¨ve CD4T cells isolated from the blood of HIV-negative donors and a single-round infectious HIV molecular clone [1]. This model was subsequently modified to include replication competent viruses and antiretroviral therapy (ART) [7, 8]. This modification was introduced for two main reasons. First, the generation of replication-deficient HIV using env deleted HIV molecular clones pseudotyped with HIV env genes can raise to the generation of replication competent viruses that can bias the results [9]. Second, the use of ART in vitro can mimic the presence of ART in HIV-infected patients. This model has now been extensively used and characterized by our group and others. Its applications include the identification and characterization of Latency-Reversing Agents (LRAs) [10–15]; to identify mechanisms controlling HIV persistence in CD4T cells

Guido Poli et al. (eds.), HIV Reservoirs: Methods and Protocols, Methods in Molecular Biology, vol. 2407, https://doi.org/10.1007/978-1-0716-1871-4_4, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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including p53, homeostatic proliferation, long noncoding RNA, and others cellular pathways [16–25]; or to study HIV integration, integration sites and clonal expansion [26–28]. Variations of this model can be used to understand whether CD4T effector function could influence HIV latency [1, 29, 30]. Finally, this model allows for the generation of latently infected cells using naı¨ve CD4T cells isolated from aviremic participants to study CD8T cell effector function [31]. Here, we describe in detail the generation of latently infected central memory CD4 T cells using naı¨ve CD4T cells isolated from peripheral blood.

2

Materials All the solutions prepared must be and remain sterile throughout the procedure.

2.1 Media, Cytokines and Antiretroviral Drugs

1. Cell media: RPMI supplemented with 2 mM L-Glutamine, 50,000 U of Penicillin–Streptomycin and 10% Fetal Bovine Serum (see Note 1). Store at 4  C. 2. Dynabeads Human T-Activator CD3/CD28: magnetic beads coated with antibodies against human CD3 and CD28. 3. 50 μg/mL TGF-β (see Note 2). Prepare working concentration at 500 ng/mL in cell media. Aliquot stock and working concentration and store at 20  C. 4. 200 μg/mL anti-human IL-4 and anti-human IL-12 (see Note 2). Aliquot and store at 20  C. 5. 106 IU/mL IL-2 (see Note 2). Aliquot and store at 6. 10 mM Raltegravir. Aliquot and store at 7. 10 mM Nelfinavir. Aliquot and store at

2.2 Cell Purification and Staining

20  C.

20  C. 20  C.

1. Density gradient medium: 1.077 g/mL density sterile medium such as Ficoll-Paque or Lymphoprep. Store at 4  C. 2. Blood buffer: 2 mM EDTA and 2% FBS in PBS. Store at 4  C. 3. Human Naı¨ve CD4 negative selection kit: kit for the isolation of untouched human naı¨ve CD4 T cells such as EasySep™ Human Naı¨ve CD4+ T Cell Isolation Kit or Miltenyi Naive CD4+ T Cell Isolation Kit II. It should contain antibodies against CD8, CD14, CD16, CD19, CD20, CD25, CD36, CD56, CD61, CD66b, CD123, HLA-DR, TCRγ/δ, and glycophorin A. Store at 4  C. 4. Staining buffer: 3% FBS in PBS. Store at 4  C. 5. Anti-human CD4 antibody clone S3.5 conjugate to allophycocyanin (APC). Store at 4  C.

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6. Anti-HIV Gag antibody clone KC57 conjugated to fluorescein isothiocyanate (FITC). Store at 4  C. 7. Fixation and permeabilization buffer such as BD Cytofix/ Cytoperm™. Store at 4  C. 8. Washing buffer such as BD Perm/Wash™. Store at 4  C. 9. 2% PFA: 2% Paraformaldehyde in PBS. Store at 4  C. 2.3

Tissue Culture

1. Sterile 96 well-round plates suitable for tissue culture. 2. Tissue culture flask and plates of different sizes. 3. Sterile reservoirs of different volumes. 4. 12-channel 300 μL multichannel pipette.

3

Methods All the procedures have to be performed under sterile conditions in a biological safety cabinet wearing the appropriate personal protection equipment required to work with HIV. Cells are maintained in a cell culture incubator at 37  C and 5% CO2.

3.1 PBMC Isolation and Naı¨ve CD4T Purification Using Density Gradient Centrifugation

1. Set out blood buffer and density gradient medium from the fridge the night before isolation to allow them to reach room temperature. 2. Dilute blood 1:1 with blood buffer (see Note 3). 3. Prepare 50 mL conical centrifuge tubes by adding 20 mL of density gradient medium into each tube (see Note 4). 4. Add diluted blood to an even number of 50 mL conical centrifuge tubes up to 50 mL. 5. Spin at room temperature for 30 min at 258  g with the breaks off (see Note 4). 6. Transfer the PBMC-containing interface layer into new 50 mL conical centrifuge tubes. 7. If needed, add blood buffer to bring total volume of each 50 mL conical centrifuge tube to 40 mL to help wash the cells. 8. Spin at 4  C for 5 min at 525  g. Discard supernatant. 9. Resuspend each pellet in small amount of blood buffer and transfer each suspension to a single 50 mL conical centrifuge tube. 10. Fill the 50 mL conical centrifuge tube to 40 mL with blood buffer to wash one more time. 11. Spin at 4  C for 5 min at 525  g and discard the supernatant. Resuspend pelleted PBMCs in 30 mL of cell media and count cells.

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12. Isolate naı¨ve CD4T cells using commercially available negative selection kits following manufacture indications (see Note 5). 3.2 Generation of Latently Infected TCM Cells

1. At Day 0, prepare naı¨ve CD4T cells in cell media at a density of 0.5  106 cells per mL with 2 μg/mL anti-human IL-12, 1 μg/ mL anti-human IL-4, and 10 ng/mL of TGF-β (see Note 6). 2. Add Dynabeads Human T-Activator CD3/CD28 at 1 magnetic bead per cell (see Note 7). 3. Plate cells in sterile 96-well round plates at 100 μL per well and culture for 72 h in a cell incubator (see Note 8). 4. After activation (Day 3), resuspend each well to break cell clumps and transfer the cells to a sterile centrifuge tube. Place the tube against a magnetic field for 1–2 min. Carefully, transfer the cells without touching the beads attracted to the magnetic field to a clean and sterile centrifuge tube. 5. Count the cells and centrifuge at 525  g for 5 min. Aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL of IL-2 (see Note 9). Culture for 24 h in a cell incubator. 6. Next day (Day 4), count the cells, transfer to a clean and sterile centrifuge tube and centrifuge at 525  g for 5 min at room temperature. Aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL of IL-2 (see Note 10). Culture for 24 h in a cell incubator. 7. Next day (Day 5), count the cells, transfer to a clean and sterile centrifuge tube and centrifuge at 525  g for 5 min at room temperature. Aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL of IL-2. Culture for 48 h in a cell incubator. 8. At day 7, cells are ready to infect. Count the cells and divide the culture into two parts (1/4th of the culture as Part I and 3/4ths of the culture as Part II). 9. Part I. This is your uninfected fraction to use as controls in experiments and/or gating controls for the p24 stain (see protocol below). Centrifuge at 525  g for 5 min at room temperature. Aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL of IL-2. Culture for 72 h in a cell incubator (see Note 11). 10. Part II. This is your infected fraction. This is further divided into two parts (3/4ths as Part IIA and 1/4th as Part IIB). 11. Part IIA. Centrifuge the culture at 525  g for 5 min at room temperature. Aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL of IL-2.

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12. Part IIB. Centrifuge at 525  g for 5 min at room temperature. Aspirate media and resuspend at 106 cells per mL in media containing NL43 HIV virus at a multiplicity of infection of 1 (see Note 12). Transfer to sterile 5 mL round-bottom polystyrene tubes and centrifuge for 2 h at 1100  g at 37  C (see Note 13). 13. After infection, aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL of IL-2. Combine Part IIB with Part IIA. Culture for 72 h in a cell incubator. 14. At day 10, count uninfected and infected cultures. Take aside 105 to 2.5  105 cells to assess levels of infection by measuring the intracellular expression of HIV p24Gag (see protocol below) (see Note 14). Centrifuge at 525  g for 5 min at room temperature. Aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL of IL-2. Place the cells in a sterile reservoir, then plate cells in 96 wellround plates using a 12-channel pipette at 100 μL per well and culture for 72 h in a cell incubator (see Note 15). 15. At day 13, resuspend each well to break cell clumps and transfer the cells from the 96-well plate using a 12-channel pipette and a sterile reservoir, then transfer to a sterile centrifuge tube. Count uninfected and infected cultures and take aside 105 to 2.5  105 cells to assess levels of infection (see Note 16). Centrifuge at 525  g for 5 min at room temperature. Aspirate media and resuspend at 106 cells per mL in cell media supplemented with 30 IU/mL IL-2 and supplemented with 1 μM Raltegravir and 0.5 μM Nelfinavir (see Note 17). Culture for 96 h in a cell incubator. 16. At day 17, latently infected cells are isolated using magnetic beads coated with antibodies against human CD4 (see Note 18). 17. After isolation, latently infected cells can be reactivated using the desired LRA or used for any other analysis (see Note 19). 3.3 Staining for Surface CD4 and Intracellular HIV Gag

1. Take between 105 and 2.5  105 cells and transfer to a 5 mL round-bottom polystyrene tube. Add 1 mL of PBS and centrifuge for 5 min at 525  g at 4  C (see Note 20). 2. Aspirate supernatant and add 100 μL of PBS containing a viability dye. Vortex and incubate for 10 min at 4  C (see Note 21). 3. Add 1 mL of PBS and centrifuge for 5 min at 525  g at 4  C. 4. Aspirate supernatant and add 100 μL of staining buffer containing anti-human CD4 antibody clone S3.5 conjugate to allophycocyanin (APC). Vortex and incubate for 30 min at 4  C (see Note 22).

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5. Add 1 mL of staining buffer and centrifuge for 5 min at 300  g at 4  C. 6. Aspirate supernatant and add 100 μL of fixation and permeabilization buffer. Vortex and incubate for 30 min at 4  C (see Note 23). 7. Add 1 mL of washing buffer and centrifuge for 5 min at 525  g at 4  C (see Note 24). 8. Aspirate supernatant and add 100 μL washing buffer containing anti-HIV Gag antibody clone KC57 conjugated to fluorescein isothiocyanate (FITC). Vortex and incubate for 30 min at 4  C (see Note 22). 9. Add 1 mL of washing buffer and centrifuge for 5 min at 300  g. 10. Aspirate supernatant and add 100 to 200 μL of 2% PFA. Keep at 4  C, in the dark, until analyzing by flow cytometry.

4

Notes 1. Fetal calf serum or human serum can also be used. Avoid using advance medias that do not require serum. 2. Reconstitute as indicated by the manufacturer. Make aliquots to avoid multiple freezing-thawing cycles. 3. This protocol has been optimized for buffy coats. 4. If using SepMate™ tubes, add only 15 mL of Lymphoprep™ to the SepMate™ tube and centrifuge at room temperature for 10 min at 1200  g leaving the breaks on in the centrifuge. All relative centrifugal forces provided are for the average radius of a TX-400 4  400 mL Swinging Bucket Rotor (Thermo Scientific™). 5. If using frozen PBMCs, thaw cells the day before isolation and keep in culture at 37  C overnight at 1–5 million cells per mL in cell media. 6. We have estimated that activating one million naı¨ve CD4 T cell can render approximately one million latently infected central memory CD4 T cell at day 17 following this protocol. 7. The protocol has been optimized to use human T-Activator CD3/CD28 Dynabeads. Other alternatives to activate naı¨ve CD4T cells have not been explored. 8. After 72 h, a white pellet of activated CD4T cells with the magnetic beads should be apparent at the bottom of each well. This indicates that the cells have been properly activated and have started to proliferate.

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1000 Uninfected Cultures Infected Cultures

nt/n0

100

10

1

0

3

6

9

12

15

18

Days

Fig. 1 Proliferation curves after activation and infection of naı¨ve CD4T cells from 23 donors (12 female and 11 male)

9. IL-2 should be added fresh to the cell media each time. Different size tissue culture plates or flasks can be used at this time. We recommend to use 24-well plates for up to 1 mL, 12-well plate for 1–2 mL, 6-well plate for 2–4 mL, T25 flask for 5–10 mL, T75 flask for 10–50 mL and T175 flask for 50–150 mL. We recommend IL-2 available from the NCI or any other IL-2 that promotes strong proliferation. See Fig. 1 for a proliferation curve of the uninfected and infected cultures of 23 donors. 10. During the first days after activation, CD4T cells are exponentially growing and rapidly consuming the media. When media is not changed at day 4, cells can overgrow and die. This hinders the generation of latency infected cells. 11. We recommend setting aside uninfected cells to use as controls for the different experiments. 12. Multiplicity of infection should be determined either in the cell line SupT1 or in activated CD4T cells at day 7. This protocol has been optimized for the lab adapted strain of HIV NL4-3. 13. Most centrifuges do not have the possibility to warm up. To achieve 37  C, use a refrigerated centrifuge and set up the temperature to 37  C. Run the centrifuge for 30 min at 1800  g or at 90% of maximum allowable spin prior to spin infection to warm the centrifuge to the indicated temperature. 14. You want to achieve 5–8% of the cells infected determined by the expression of HIV p24Gag and the downregulation of CD4 [8].

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15. This process, termed crowding, allows for an increase in cellto-cell HIV transmission and allows for a higher efficiency on the generation of latently infected cells. Up to 250 μL can be used per well without apparent loss of viability or infectivity. 16. Levels of infection should have increased relative to day 10. The average increase in p24 positive cells is four to fivefold [8]. 17. Other antiretrovirals can be used. To assess that the antiretroviral regimen selected is appropriate, we suggest to culture cells during the crowding phase in the presence of the different antiretrovirals to ensure that cell-to-cell transmission is blocked. 18. We define latently infected cells to those cells that are positive for the expression of CD4 and negative for the expression of p24Gag. HIV downregulates CD4 from the surface of T cells [32, 33]. We use a positive selection kit to eliminate any residual productively infected cell as well as any CD4 negative cell. We have used Dynabeads® CD4 positive isolation kit. This kit also allows for the detachment of both the antibody and the bead from the latently infected cells. We use the recommended manufacture protocol with some variations. 75 μL instead of 25 μL of the magnetic bead suspension is added per 107 cells to increase the efficiency of the recovery. We use double amount of Buffer II per 107 cells to increase recovery and cell are washed twice in order to remove any residual DETACHaBead solution that can affect further analysis. 19. For LRA assays, we recommend keeping the cells in the presence of 30 IU/mL of IL-2 to increased survival and ART to avoid ongoing replication. Maximal stimulation with antihuman CD3 and anti-human CD28 antibodies peaks at 48 h post-stimulation. 20. We recommend using sterile PBS with no azide. 21. The viability dye has to be compatible with fixation and permeabilization protocols as well as compatible with the fluorochromes selected for staining. Incubation of the viability dye has to be done in a 4  C fridge and not on ice or as recommended by the manufacture. 22. We recommend titrating the antibody using primary CD4 T cells to identify the proper amount of antibody to use for the staining. Incubation of the antibody has to be done in a 4  C fridge and not on ice. At this point, the staining can be stopped, placing the cells in 2% PFA until the following or next day. Wash with PBS and continue with the intracellular stain.

The Cultured TCM Model of HIV Latency

53

23. We recommend the BD Cytofix/Cytoperm™ solution. Incubation of the buffer has to be done in a 4  C fridge and not on ice. 24. We recommend the BD Perm/Wash™ buffer solution.

Acknowledgments I would like to thank Indra Sarabia and Amanda B. Macedo for their input in the writing and proofreading of the manuscript. The work in A.B.’s lab is currently supported by the National Institute of Allergy and Infectious Diseases and National Institute of Health grants R01-AI124722, R21/R33-AI116212, R01-AI147845, UM1-AI126617, and P30-AI117970. References 1. Bosque A, Planelles V (2009) Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells. Blood 113(1):58–65. https://doi.org/10.1182/blood-200807-168393 2. Saleh S, Solomon A, Wightman F, Xhilaga M, Cameron PU, Lewin SR (2007) CCR7 ligands CCL19 and CCL21 increase permissiveness of resting memory CD4+ T cells to HIV-1 infection: a novel model of HIV-1 latency. Blood 110(13):4161–4164. https://doi.org/10. 1182/blood-2007-06-097907 3. Yang HC, Xing S, Shan L, O’Connell K, Dinoso J, Shen A, Zhou Y, Shrum CK, Han Y, Liu JO, Zhang H, Margolick JB, Siliciano RF (2009) Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation. J Clin Invest 119(11):3473–3486. https://doi.org/10. 1172/JCI39199 4. Marini A, Harper JM, Romerio F (2008) An in vitro system to model the establishment and reactivation of HIV-1 latency. J Immunol 181 (11):7713–7720 5. Tyagi M, Pearson RJ, Karn J (2010) Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol 84 (13):6425–6437. https://doi.org/10.1128/ JVI.01519-09 6. Lassen KG, Hebbeler AM, Bhattacharyya D, Lobritz MA, Greene WC (2012) A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs. PLoS One 7(1):e30176. https://doi.org/10.1371/ journal.pone.0030176

7. Martins LJ, Bonczkowski P, Spivak AM, De Spiegelaere W, Novis CL, DePaula-Silva AB, Malatinkova E, Trypsteen W, Bosque A, Vanderkerckhove L, Planelles V (2016) Modeling HIV-1 latency in primary T cells using a replication-competent virus. AIDS Res Hum Retrovir 32(2):187–193. https://doi.org/10. 1089/aid.2015.0106 8. Macedo AB, Resop RS, Martins LJ, Szaniawski MA, Sorensen ES, Spivak AM, Nixon DF, Jones RB, Planelles V, Bosque A (2018) Influence of biological sex, age and HIV status in an in vitro primary cell model of HIV latency using a CXCR4 tropic virus. AIDS Res Hum Retrovir 34(9):769–777. https://doi.org/10. 1089/AID.2018.0098 9. Bonczkowski P, De Spiegelaere W, Bosque A, White CH, Van Nuffel A, Malatinkova E, Kiselinova M, Trypsteen W, Witkowski W, Vermeire J, Verhasselt B, Martins L, Woelk CH, Planelles V, Vandekerckhove L (2014) Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs. Retrovirology 11:70. https://doi.org/10.1186/s12977014-0070-3 10. Novis CL, Archin NM, Buzon MJ, Verdin E, Round JL, Lichterfeld M, Margolis DM, Planelles V, Bosque A (2013) Reactivation of latent HIV-1 in central memory CD4(+) T cells through TLR-1/2 stimulation. Retrovirology 10:119. https://doi.org/10.1186/17424690-10-119 11. Bosque A, Nilson KA, Macedo AB, Spivak AM, Archin NM, Van Wagoner RM, Martins LJ, Novis CL, Szaniawski MA, Ireland CM, Margolis DM, Price DH, Planelles V (2017)

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Benzotriazoles reactivate latent HIV-1 through inactivation of STAT5 SUMOylation. Cell Rep 18(5):1324–1334. https://doi.org/ 10.1016/j.celrep.2017.01.022 12. Macedo AB, Novis CL, De Assis CM, Sorensen ES, Moszczynski P, Huang SH, Ren Y, Spivak AM, Jones RB, Planelles V, Bosque A (2018) Dual TLR2 and TLR7 agonists as HIV latencyreversing agents. JCI Insight 3(19):e122673. https://doi.org/10.1172/jci.insight.122673 13. Spina CA, Anderson J, Archin NM, Bosque A, Chan J, Famiglietti M, Greene WC, Kashuba A, Lewin SR, Margolis DM, Mau M, Ruelas D, Saleh S, Shirakawa K, Siliciano RF, Singhania A, Soto PC, Terry VH, Verdin E, Woelk C, Wooden S, Xing S, Planelles V (2013) An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 9(12):e1003834. https://doi.org/10.1371/journal.ppat. 1003834 14. Duverger A, Wolschendorf F, Anderson JC, Wagner F, Bosque A, Shishido T, Jones J, Planelles V, Willey C, Cron RQ, Kutsch O (2014) Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation. J Virol 88(1):364–376. https://doi.org/10.1128/JVI.02682-13 15. Larson EC, Novis CL, Martins LJ, Macedo AB, Kimball KE, Bosque A, Planelles V, Barrows LR (2017) Mycobacterium tuberculosis reactivates latent HIV-1 in T cells in vitro. PLoS One 12 (9):e0185162. https://doi.org/10. 1371/journal.pone.0185162 16. White CH, Moesker B, Beliakova-Bethell N, Martins LJ, Spina CA, Margolis DM, Richman DD, Planelles V, Bosque A, Woelk CH (2016) Transcriptomic analysis implicates the p53 signaling pathway in the establishment of HIV-1 latency in central memory CD4 T cells in an in vitro model. PLoS Pathog 12(11): e1006026. https://doi.org/10.1371/journal. ppat.1006026 17. Wolschendorf F, Bosque A, Shishido T, Duverger A, Jones J, Planelles V, Kutsch O (2012) Kinase control prevents HIV-1 reactivation in spite of high levels of induced NF-kappaB activity. J Virol 86(8):4548–4558. https://doi.org/10.1128/JVI.06726-11 18. Budhiraja S, Famiglietti M, Bosque A, Planelles V, Rice AP (2013) Cyclin T1 and CDK9 T-loop phosphorylation are downregulated during establishment of HIV-1 latency in primary resting memory CD4+ T cells. J Virol 87(2):1211–1220. https://doi.org/10.1128/ JVI.02413-12

19. Gavegnano C, Detorio M, Montero C, Bosque A, Planelles V, Schinazi RF (2014) Ruxolitinib and tofacitinib are potent and selective inhibitors of HIV-1 replication and virus reactivation in vitro. Antimicrob Agents Chemother 58(4):1977–1986. https://doi. org/10.1128/AAC.02496-13 20. Saayman S, Ackley A, Turner AW, Famiglietti M, Bosque A, Clemson M, Planelles V, Morris KV (2014) An HIV-encoded antisense long noncoding RNA epigenetically regulates viral transcription. Mol Ther 22(6):1164–1175. https://doi.org/10. 1038/mt.2014.29 21. Trypsteen W, White CH, Mukim A, Spina CA, De Spiegelaere W, Lefever S, Planelles V, Bosque A, Woelk CH, Vandekerckhove L, Beliakova-Bethell N (2019) Long non-coding RNAs and latent HIV - a search for novel targets for latency reversal. PLoS One 14(11): e0224879. https://doi.org/10.1371/journal. pone.0224879 22. Murry JP, Godoy J, Mukim A, Swann J, Bruce JW, Ahlquist P, Bosque A, Planelles V, Spina CA, Young JA (2014) Sulfonation pathway inhibitors block reactivation of latent HIV-1. Virology 471-473:1–12. https://doi.org/10. 1016/j.virol.2014.08.016 23. Bosque A, Famiglietti M, Weyrich AS, Goulston C, Planelles V (2011) Homeostatic proliferation fails to efficiently reactivate HIV-1 latently infected central memory CD4+ T cells. PLoS Pathog 7(10):e1002288. https://doi. org/10.1371/journal.ppat.1002288 24. Ren Y, Huang SH, Patel S, Conce Alberto WD, Magat D, Ahimovic DJ, Macedo AB, Durga R, Chan D, Zale E, Mota TM, Truong R, Rohwetter T, McCann CD, Kovacs CM, Benko E, Wimpelberg A, Cannon CM, Hardy WD, Bosque A, Bollard CM, Jones RB (2020) BCL-2 antagonism sensitizes cytotoxic t cell-resistant hiv reservoirs to elimination ex vivo. J Clin Invest 130(5):2542–2559. https://doi.org/10.1172/JCI132374 25. Huang SH, Ren Y, Thomas AS, Chan D, Mueller S, Ward AR, Patel S, Bollard CM, Cruz CR, Karandish S, Truong R, Macedo AB, Bosque A, Kovacs C, Benko E, Piechocka-Trocha A, Wong H, Jeng E, Nixon DF, Ho YC, Siliciano RF, Walker BD, Jones RB (2018) Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells. J Clin Invest 128(2):876–889. https://doi. org/10.1172/JCI97555 26. Sunshine S, Kirchner R, Amr SS, Mansur L, Shakhbatyan R, Kim M, Bosque A, Siliciano RF, Planelles V, Hofmann O, Ho Sui S, Li JZ (2016) HIV integration site analysis of cellular

The Cultured TCM Model of HIV Latency models of HIV latency with a probe-enriched next-generation sequencing assay. J Virol 90 (9):4511–4519. https://doi.org/10.1128/ JVI.01617-15 27. Lusic M, Marini B, Ali H, Lucic B, Luzzati R, Giacca M (2013) Proximity to PML nuclear bodies regulates HIV-1 latency in CD4+ T cells. Cell Host Microbe 13(6):665–677. https://doi.org/10.1016/j.chom.2013.05. 006 28. Sherrill-Mix S, Lewinski MK, Famiglietti M, Bosque A, Malani N, Ocwieja KE, Berry CC, Looney D, Shan L, Agosto LM, Pace MJ, Siliciano RF, O’Doherty U, Guatelli J, Planelles V, Bushman FD (2013) HIV latency and integration site placement in five cell-based models. Retrovirology 10:90. https://doi.org/10. 1186/1742-4690-10-90 29. Nguyen K, Das B, Dobrowolski C, Karn J (2017) Multiple histone lysine methyltransferases are required for the establishment and maintenance of HIV-1 latency. MBio 8(1): e00133-17. https://doi.org/10.1128/mBio. 00133-17

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30. Dobrowolski C, Valadkhan S, Graham AC, Shukla M, Ciuffi A, Telenti A, Karn J (2019) Entry of polarized effector cells into quiescence forces HIV latency. MBio 10(2):e00337-19. https://doi.org/10.1128/mBio.00337-19 31. Thomas AS, Jones KL, Gandhi RT, McMahon DK, Cyktor JC, Chan D, Huang SH, Truong R, Bosque A, Macedo AB, Kovacs C, Benko E, Eron JJ, Bosch RJ, Lalama CM, Simmens S, Walker BD, Mellors JW, Jones RB (2017) T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy. PLoS Pathog 13(9):e1006629. https://doi. org/10.1371/journal.ppat.1006629 32. Garcia JV, Miller AD (1991) Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350 (6318):508–511. https://doi.org/10.1038/ 350508a0 33. Willey RL, Maldarelli F, Martin MA, Strebel K (1992) Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol 66(12):7193–7200

Chapter 5 A Reliable Primary Cell Model for HIV Latency: The QUECEL (Quiescent Effector Cell Latency) Method Meenakshi Shukla, Fredrick Kizito, Uri Mbonye, Kien Nguyen, Curtis Dobrowolski, and Jonathan Karn Abstract One of the main methods to generate the HIV reservoir is during the transition of infected activated effector CD4 T cells to a memory phenotype. The QUECEL (Quiescent Effector Cell Latency) protocol mimics this process efficiently and allows for production of large numbers of latently infected CD4+ T cells. After polarization and expansion, CD4+ T cells are infected with a single round reporter virus which expressed GFP/CD8a. The infected cells are purified and coerced into quiescence using a defined cocktail of cytokines including TGF-β, IL-10, and IL-8, producing a homogeneous population of latently infected cells. Since homogeneous populations of latently infected cells can be recovered, the QUECEL model has an excellent signal-to-noise ratio, and has been extremely consistent and reproducible in numerous experiments performed during the last 5 years. The ease, efficiency, and accurate mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency. Key words HIV latency, Memory T-cells, QUECEL model, Cytokines, Cellular quiescence

1

Introduction HIV persists due to a pool of transcriptionally silenced, but replication-competent proviruses, found in a small population of resting memory CD4+ T cells (1 to 100 per 106 cells) accumulating in the peripheral blood [1] and tissues [2]. Since silenced proviruses produce only minimal viral RNA and proteins, they are refractory to antiviral drugs and effectively evade immune surveillance. Our understanding of HIV latency and persistence has been complicated by the small numbers of latently infected cells found in the circulation, the difficulty of obtaining comprehensive sets of tissue samples from patients, the lack of known phenotypic markers that can distinguish latently infected cells from uninfected ones, and limited information about the behavior of tissue reservoirs in vivo. Contemporary HIV research has therefore been propelled

Guido Poli et al. (eds.), HIV Reservoirs: Methods and Protocols, Methods in Molecular Biology, vol. 2407, https://doi.org/10.1007/978-1-0716-1871-4_5, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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by the development of models of T cell latency that can be implemented in the laboratory setting. Although latency can be established in transformed T cell lines, such as Jurkat T-cells [3–7], this can lead to potentially unrepresentative mechanisms of HIV latency since the host cell is not quiescent [7–9]. A more physiologically relevant approach is to infect primary cells isolated from healthy donors. In many instances, HIV latency is established during the transition of effector memory cells to a quiescent memory cell phenotype [10–12]. Primary cell models of HIV latency [13] therefore typically involve infection of an activated cell population that is then allowed to enter quiescence [14–16]. Alternatively, resting cells can be infected directly [12, 17, 18]. Unfortunately, most primary cell models for HIV latency correlate poorly with the reactivation behavior of patient cells [7, 19]. The QUECEL model [8, 19–21], which is described in detail in this chapter, is a refinement of the model of Bosque and Planelles [14, 22]. Briefly, Naı¨ve helper T cells are polarized into the four major effector T cell subsets (Th1, Th2, Th17, and Treg). The Th17 polarization phenotype, which we routinely use since these cells tend to retain a higher degree of viability than the other polarization phenotypes, represents the most abundant effector T cell population in the lamina propria of the GI tract [23–25]. QUECEL generates a large and homogeneous population of latently infected CD4+ memory cells. By purifying HIV infected cells and inducing cell quiescence with a defined cocktail of cytokines, we have eliminated the largest problems with previous primary cell models of HIV latency: variable infection levels, ill-defined polarization states, and inefficient shut down of cellular transcription. This scalable and highly reproducible model of HIV latency therefore permits a detailed analysis of cellular mechanisms controlling HIV latency and reactivation and we have used it successfully to study the epigenetic, transcriptomic, and cell biological underpinnings of HIV latency.

2

Materials

2.1 Cytokines and Antibodies for Generating Effector CD4 T Cells

1. IL-23: stock concentration 50 μg/ml and final working concentration 50 ng/ml; TGF-β: stock concentration 5 μg/ml and final working concentration 5 ng/ml; IL-1β: stock concentration 10 μg/ml and final working concentration 10 ng/ml; IL-6: stock concentration 10 μg/ml and final working concentration 10 ng/ml; IL-7: stock concentration 10 μg/ml and final working concentration 10 ng/ml; IL-2: stock concentration 60,000 IU/ml and final working concentration 60 IU/ ml. All cytokines are reconstituted in PBS containing 0.2% BSA and stored at
20  C until use.

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59

2. Anti-Human IL-4: stock concentration 500 μg/ml and final working concentration 500 ng/ml; anti-Human IFN-γ: stock concentration 10 μg/ml and final working concentration 10 ng/ml. Both antibodies are reconstituted in cell culture grade water and stored at +4  C until use. 2.2 Cell Growth Media

RPMI media, 10% FBS, 100 μg/ml Primocin (stock concentration 50 mg/ml) and 25 mM HEPES (stock concentration 1 M) together with their required cytokines as described in tables.

2.3 Source of Cells and Virus

Frozen PBMCs from healthy donors. VSV glycoproteinpseudotyped HIV-1 pNL4-3 delta gag CD8a/GFP-IRES-Nef reporter virus used at multiplicity of infection (MOI) of 2.0 (at 5  106 cells per ml).

2.4

Human Naı¨ve CD4+ T Cell enrichment kit and Mouse CD8a isolation kit available from various suppliers such as STEMCELL Technologies.

Cell Isolation Kit

2.5 Cell Polarization and Reactivation Reagents 2.6 Tissue Culture Supplies and Instrument

10 μg/ml concanavalin A (ConA) (stock concentration 10 mg/ml) for cell polarization, and 25 μl/ml Dynabeads Human T-Activator CD3/CD28 for cell reactivation. 1. 50 ml and 17 ml conical tube; T25 and T75 tissue culture flasks; 96 well U-bottom plates; 10 cm plates, 5 ml and 25 ml pipettes; 1000 μl, 200 μl, 20 μl, and 10 μl Barrier tips. 2. Centrifuge and RoboSep™ Fully Automated Cell Separator.

2.7 Flow Antibody and Reagents

2.8 RNA FISH Reagents and Supplies

For Flow analysis AF647-HIV EH1 Nef (Jim Hoxie laboratory) antibody, formaldehyde (Electron Microscope Sciences) and Permeabilization Buffer (eBioscience). 1. Custom Stellaris® RNA FISH probes (Biosearch Technologies) 18 to 20-nucleotides and labeled at the 50 end with either a 6-carboxytetramethylrhodamine (TAMRA) or Quasar 670® fluorophores. 2. 20 Saline-sodium citrate (SSC) buffer (3 M sodium chloride and 300 mM trisodium citrate; adjusted to pH 7.0 with HCl). 3. RNA FISH Wash Buffer A: Stellaris® RNA FISH Wash Buffer A (Biosearch Technologies) or 2 SSC, 10% de-ionized formamide. 4. RNA FISH Hybridization Buffer: 10% dextran sulfate, 2 SSC, 10% deionized formamide. 5. Stellaris® RNA FISH Wash Buffer B (Biosearch Technologies) or 2 SSC.

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6. Prolong Diamond Antifade mountant (Life Technologies). 7. 4,6-Diamidino-2-phenylindole (DAPI) dye (1 μg/ml).

3

Methods An outline of the QUECEL method is shown in Fig. 1. Naı¨ve T-cells are polarized and after 6 days of expansion the replicating cells are infected using a single round VSV pseudotyped reporter virus that expresses GFP/CD8a (Fig. 1a) [3, 4, 26]. The infected cells are then purified by magnetic bead isolation and coerced into quiescence using a defined cocktail of cytokines including TGF-β, IL-10, and IL-8, producing a homogeneous population of latently infected cells (Fig. 1b). Flow cytometry using the cell cycle markers demonstrated that the cells maintained the correct polarization phenotype and had withdrawn from the cell cycle [19]. Both GFP and HIV nef expression can be used to monitor the fraction of infected cells and entry of cells into quiescence (Fig. 1c). Once the cells have entered quiescence, the level of HIV Nef protein expression is reduced to almost undetectable levels (1%), which is indicative of HIV latency. GFP levels also decline, but because of the high stability of the CD8a-GFP fusion protein, a moderate level of GFP persists in the quiescent cells. Upon stimulation through the TCR (α-CD3/ α-CD28 Dynal magnetic beads), the fraction of cells expressing Nef increases dramatically (>82%) and there is a concomitant increase in GFP levels (Fig. 1c). The QUECEL model is extremely robust and shows minimal variation between replicate samples and between donors in assays performed over a period of more than 2 years (Fig. 1d). RNA FISH and immunofluorescence can also be used to show that the latently infected cells produce minimal levels of HIV RNA and the HIV Tat transactivator protein, Tat (Fig. 1e). We also routinely monitor cells using an antibody to CDK9 pSer175 as a marker for transcriptionally active P-TEFb [27] since there is a strict correlation between cells that have activated P-TEFb and cells that produce HIV proteins. An improvement to the original published QUECEL method [19], which is described here, is the addition of IL-7 to improve cell viability. The ease, efficiency, and accurate mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.

3.1 Day 0: Activation and Initial Polarization

1. Isolate naı¨ve CD4 T cells using Human Naı¨ve CD4+ T Cell Enrichment Kit following the manufacturer’s directions. Typically, 50  106 PBMCs yield 5  106 naı¨ve CD4 T cells. Resuspend the naı¨ve CD4 T cells at 5  105 cells per ml, which is approximately 10 ml of media per 50  106 PBMCs

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Fig. 1 Ex vivo QUECEL model for HIV latency. (a) Structure of lentiviral reporter virus. (b) Time line for polarization and infection of naı¨ve CD4+ T cells. In this example cells are polarized to a Th17 phenotype. After infection, cells are placed in media containing a limiting amount of cytokines to allow the cells to return to a resting state. (c) Flow cytometric analysis of the expression of the reporter genes GFP (vertical) and Nef (horizontal) after infection, after isolation of infected cells, after entering quiescence and after restimulation through the TCR. (d) Reproducibility of the QUECEL assay. (e) Induction of HIV RNA and 7SK snRNA after reactivation of latent proviruses by T-cell receptor activation of quiescent Th17 cells (Top) and immunofluorescence imaging of HIV Tat. Images are taken at 100 using a high resolution DeltaVision deconvolution microscope. Scale bars represent a length of 10 μm and the images have been enlarged three times

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Table 1 Polarization Cytokines Subtype

Cytokine or antibody

Stock Conc.

Dilution

Source

Catalog No.

Th1

Anti-human IFN-γ

10 μg/ml

1:1000

PeproTech

500-M90

Anti-human IL-4

500 μg/ml

1:1000

PeproTech

500-M04

Anti-human IL-4

500 μg/ml

1:1000

PeproTech

500-M04

Anti-human IFN-γ

10 μg/ml

1:1000

PeproTech

500-M90

TGF-β

5 μg/ml

1:1000

PeproTech

100-21C

Anti-human IL-4

500 μg/ml

1:1000

PeproTech

500-M04

Anti-human IFN-γ

10 μg/ml

1:1000

PeproTech

500-M90

IL-1β

10 μg/ml

1:1000

PeproTech

200-01B

IL-6

30 μg/ml

1:1000

PeproTech

200-06

IL-23

50 μg/ml

1:1000

PeproTech

200-23

TGF-β

5 μg/ml

1:1000

PeproTech

100-21C

Anti-human IL-4

500 μg/ml

1:1000

PeproTech

500-M04

Anti-human IFN-γ

10 μg/ml

1:1000

PeproTech

500-M90

Anti-human IL-12

500 μg/ml

1:1000

PeproTech

500-M12

Th2

Th17

TReg

used. 5  106 of polarized cells yield 50–100  106 effector cells after the expansion phase of the protocol. 2. Add polarization cytokines and antibodies (Table 1), together with concanavalin A to a final concentration of 10 μg/ml. 3. Distribute cells into an upright T25 flask to allow for cell-tocell contact. 4. Incubate cells for 72 h at 37  C in a CO2 incubator. 3.2 Day 3: Continuing Polarization

1. Measure out the same amount of primary cell media used at day 0, and add polarization cytokines following the dilutions shown in Table 1, together 10 μg/ml concanavalin A and 60 IU/ml of IL-2. 2. Incubate cells for 72 h at 37  C in a CO2 incubator.

3.3 Day 6: Infection with Pseudotyped Reporter Virus

Cells are ready for infection after the sixth day of polarization. Since primary cells are notoriously hard to infect, the most efficient infections are achieved by spinoculation for a long period of time using a high titer virus stock as follows: 1. Pellet the polarized cells and discard the supernatant. 2. Use the pHR0 -Nef+-CD8a/GFP virus when using the CD8a protein for purification of the infected cells. Use 1 ml of a high titer concentrated virus stock (MOI of 10 on Jurkat cells) per 10  106 polarized cells.

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Table 2 Maintenance Cytokines Subtype

Cytokine

Stock Conc.

Dilution

Source

Catalog No.

Th1, Th2, TReg

rIL-2

60,000 IU/ml

1:1000

NIH AIDS reagent program

136

Th17

rIL-2 rIL-23 IL-7

60,000 IU/ml 50 μg/ml 10 μg/ml

1:1000 1:1000 1:1000

NIH AIDS reagent program PeproTech

136 200-23 200-07

3. Dilute the virus in sufficient primary cell media and cell-specific cytokines (Table 2) to resuspend the cells at 5  106 cells per ml (i.e., for 10  106 cells, add 1 ml primary cell media to 1 ml concentrated virus and 2 μl of cytokine stock). 4. Aliquot 1 ml of cells into each well of a 24 well plate. It is convenient to split the cells evenly into two plates to provide a balance for the centrifugation. 5. Spin the plates at 2000  g for 90 min 23  C and 5 acceleration/9 deceleration. 6. Remove the cells from the centrifuge and place at 37  C in a CO2 incubator overnight. This will increase the amount of infection. 3.4 Day 7: Dilution of Virus

Cells should have grown in number after the overnight incubation. Pool the cells and replate at 1  106 cells per ml. Do not remove the virus and simply dilute the cells with fresh media until cells are at the desired concentration.

3.5 Days 8–13: Cell Husbandry and Expansion

1. Keep the cells growing by addition of normal cytokines and fresh media once the media turns yellow (or after 3 days). Maintain cell densities (see Note 1) above 1  106 cells per ml until the needed number of cells is reached. 2. Check the percentage of GFP+ cells 48 h after infection and use this to determine the number of cells expressing GFP/CD8a (Total number of cells  % GFP+ cells). Typically, approximately half of the GFP+ cells are isolated by magnetic bead purification (see Note 2).

3.6 Day 14: CD8a+ Cell Isolation

CD8a-expressing cells are isolated using the Mouse CD8a Isolation II kit as follows: 1. Resuspend cells at 10  106 cells per 100 μl of RoboSep buffer supplemented with the appropriate cytokines (Table 2). The cells are placed in a 14 ml polystyrene round bottom sterile test tube. Be careful not to get bubbles in cell suspension or on the sides of the tube, since this will make the isolation less efficient.

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2. Turn on the RoboSep and pick the first quadrant and select the first program labeled Biotin Isolation. When working with more than one type of cell/donor use one quadrant per cell type for a total of 4 different cell types/donors per isolation. When prompted enter the amount of buffer used to resuspend the cells. If there is a low frequency of infection (less than 10%) enter twice the amount of buffer, which will result in a twofold concentration of antibody and cocktail during the isolation. 3. Load the RoboSep carousel based on the directions from the machine (it will give a diagram indicating what type of tubes are required and what each tube will have at the end of the procedure). The Positive fraction tube (located in the magnet) will be the CD8a+ cells. The negative fraction tube will be a mixed population of both infected and uninfected cells, but the frequency of infected cells will be less than it was before isolation. This mixed population can be used for controls, or for additional CD8a+ cell isolations, if additional cells are required. 3.7 Days 14 to 28: Cell Quiescence

1. Vortex the positive fraction tube from the RoboSep to remove cells and beads from the side of the tube and then centrifuge and remove the RoboSep buffer. 2. Resuspend at 1  106 cells per ml, in primary cell media with the addition of the maintenance cytokine (Table 2) depending on cell type. The cells are cultured at normal levels of cytokines for 5 days without changing the media or adding any additional cytokines (see Notes 3 and 4). During this period the cells slowly use up the cytokines and cell growth slows. 3. For the cells to enter quiescence, the amount of cytokines needs to be reduced to slow growth, mimicking what happens in vivo; this is done by using the dilutions of cytokines listed in Table 3. 4. Monitor cells every 2 days by flow cytometry (Fig. 1) until there is evidence that the cells have entered quiescence (see Note 5).

3.8

RNA FISH

1. Custom Stellaris® RNA FISH probes targeting both the 50 LTR of the HIV-1 provirus are designed from the 551-bp 50 -LTR sequence of the HIV-LTR virus by using the Stellaris® FISH Probe Designer (Biosearch Technologies, Inc., Petaluma, CA) available online at www.biosearchtech.com/ stellaris. 2. Attach cells onto poly-L-lysine–coated coverslips for 10 min at 37  C, and fix using 4% Formaldehyde for 15 min at room temperature. 3. Permeabilize cells for at least 1 h in 70% ethanol at 4  C.

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Table 3 Cytokines for Quiescence Medium

Subtype Cytokine Stock Conc.

Quiescence Media

Maintenance Media

Source

Catalog No.

Th1,

rIL-2

15,000 IU/ ml

1:4000

1:4000

NIH

136

Th2,

TGF-β1

10 μg/ml

1:1000

PeproTech 100-21C

TReg,

IL-8

50 μg/ml

1:1000

PeproTech 200-08 M

IL-10

10 μg/ml

1:1000

PeproTech 200-10

rIL-7

10 μg/ml

1:2000

1:2000

PeproTech 200-07

IL-23

50 μg/ml

1:1600

1:1600

PeproTech 200-23

TGF-β

10 μg/ml

1:1000

PeproTech 100-21C

IL-8

50 μg/ml

1:1000

PeproTech 200-08M

IL-10

10 μg/ml

1:1000

PeproTech 200-10

Th17

4. After permeabilization, coverslips are rehydrated in RNA FISH Wash Buffer A for 5 min. 5. Coverslips are then hybridized overnight in a humidified chamber at 37  C in RNA FISH Hybridization Buffer containing 35–50 μl of 2.5 μM RNA FISH probes. 6. Following hybridization, cells are immersed once in RNA FISH Wash Buffer A for 30 min at room temperature. 7. Counterstain in RNA FISH wash buffer containing 1 μg/ml of 4,6-diamidino-2-phenylindole (DAPI) dye for 30 min at room temperature. 8. Cells are washed twice in RNA FISH Wash Buffer B for 5 min at room temperature and mounted using Prolong Diamond Antifade mountant. 9. High resolution images are captured with a DeltaVision fluorescence microscope in z-series (Applied Precision Imaging, GE Healthcare) that is equipped with magnification of 100 objective lenses (Olympus) and using DeltaVision immersion oil of refractive index equivalent to 1.518 Nͼ. Scatter lights are eliminated by image deconvolution using SoftWoRx 7.0.0 software package (Applied precision).

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Notes 1. After polarization, the cells must remain in the growth cytokines at the specified concentration. These cells need to be grown at high density due to autocrine production of cytokines. A concentration of 1–3  106 per ml is ideal, and the cells will stop growing at a concentration below 1  106 per ml. It is preferable not to remove any old media from these cells, and simply add fresh media and cytokines to the cells to bring them up to the ideal concentration once the media begins to turn yellow. 2. Thy1.2 can be used as a selection marker using our pHR’-Nef +-Thy1.2-T2A-GFP virus. This vector uncouples GFP from the surface protein so that GFP can be used as an HIV activation marker. Thy1.2 is also easier to purify resulting in a higher yield of HIV infected cells during isolation. To use this option, infect using the pHR0 -Nef+-Thy1.2-T2A-GFP virus in the same manner as described for the pHR0 -Nef+-CD8a/GFP virus. To select for Thy1.2 you can use the previous described protocol except use the EasySep™ Mouse CD90.2 Positive Selection Kit II (Stemcell). 3. The cells must remain in quiescence media for at least 1 week to allow the cells to fully enter quiescence. The viability of your cells drops to about 60% due to apoptosis during entry into quiescence. Entry into quiescence should be monitored by cell cycle monitoring, typically by flow cytometry for EdU incorporation and CycB1 and CycD3 levels. 4. Since the cells will continue to expand at the beginning of the cell quiescence protocol (Days 14 to 28), it is possible to start the step down phase in the middle of the expansion phase. For example, if you want the cells to expand for 7 days, place in normal media at 1  106 cells per ml, then 2 days later add more cytokine media to bring them to 1  106 cells per ml, and culture for 5 more days without changing media or cytokines to allow them to step down. 5. The addition of IL-7, as described in this protocol, increases cell viability but also partially blocks entry of cells into full quiescence. If needed, cells can be cultured for a further week in quiescence media containing 15 IU/ml IL-2 in place of IL-7.

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References 1. Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, Smith K, Lisziewicz J, Lori F, Flexner C et al (1999) Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5:512–517 2. Yukl SA, Gianella S, Sinclair E, Epling L, Li Q, Duan L, Choi AL, Girling V, Ho T, Li P et al (2010) Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis 202: 1553–1561 3. Pearson R, Kim YK, Hokello J, Lassen K, Friedman J, Tyagi M, Karn J (2008) Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J Virol 82:12291–12303 4. Jadlowsky JK, Wong JY, Graham AC, Dobrowolski C, Devor RL, Adams MD, Fujinaga K, Karn J (2014) The negative elongation factor (NELF) is required for the maintenance of proviral latency but does not induce promoter proximal pausing of RNAP II on the HIV LTR. Mol Cell Biol 34:1911–1928 5. Jordan A, Bisgrove D, Verdin E (2003) HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22: 1868–1877 6. Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC (2006) NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J 25:139–149 7. Spina CA, Anderson J, Archin NM, Bosque A, Chan J, Famiglietti M, Greene WC, Kashuba A, Lewin SR, Margolis DM et al (2013) An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 9:e1003834 8. Nguyen K, Das B, Dobrowolski C, Karn J (2017) Multiple histone lysine methyltransferases are required for the establishment and maintenance of HIV-1 latency. MBio 8: e00133-17 9. Kim YK, Mbonye U, Hokello J, Karn J (2011) T-cell receptor signaling enhances transcriptional elongation from latent HIV proviruses by activating P-TEFb through an ERK-dependent pathway. J Mol Biol 410: 896–916

10. Kulpa DA, Talla A, Brehm JH, Ribeiro SP, Yuan S, Bebin-Blackwell A-G, Miller M, Barnard R, Deeks SG, Hazuda D et al (2019) Differentiation into an effector memory phenotype potentiates HIV-1 latency reversal in CD4+ T cells. J Virol 93:e00969–e00919 11. Shan L, Deng K, Gao H, Xing S, Capoferri AA, Durand CM, Rabi SA, Laird GM, Kim M, Hosmane NN et al (2017) Transcriptional reprogramming during effector-to-memory transition renders CD4(+) T cells permissive for latent HIV-1 infection. Immunity 47: 766–775.e763 12. Cameron PU, Saleh S, Sallmann G, Solomon A, Wightman F, Evans VA, Boucher G, Haddad EK, Sekaly RP, Harman AN et al (2010) Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton. Proc Natl Acad Sci U S A 107: 16934–16939 13. Hakre S, Chavez L, Shirakawa K, Verdin E (2012) HIV latency: experimental systems and molecular models. FEMS Microbiol Rev 36:706–716 14. Bosque A, Planelles V (2008) Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells. Blood 113:58–65 15. Bosque A, Planelles V (2011) Studies of HIV-1 latency in an ex vivo model that uses primary central memory T cells. Methods 53:54–61 16. Tyagi M, Pearson RJ, Karn J (2010) Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol 84:6425–6437 17. Pace MJ, Graf EH, Agosto LM, Mexas AM, Male F, Brady T, Bushman FD, O’Doherty U (2012) Directly infected resting CD4+T cells can produce HIV gag without spreading infection in a model of HIV latency. PLoS Pathog 8: e1002818 18. Agosto LM, Herring MB, Mothes W, Henderson AJ (2018) HIV-1-infected CD4+ T cells facilitate latent infection of resting CD4+ T cells through cell-cell contact. Cell Rep 24: 2088–2100 19. Dobrowolski C, Valadkhan S, Graham AC, Shukla M, Ciuffi A, Telenti A, Karn J (2019) Entry of polarized effector cells into quiescence forces HIV latency. MBio 10:e00337-19 20. Das B, Dobrowolski C, Luttge B, Valadkhan S, Chomont N, Johnston R, Bacchetti P, Hoh R, Gandhi M, Deeks SG et al (2018) Estrogen receptor-1 is a key regulator of HIV-1 latency that imparts gender-specific restrictions on the

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latent reservoir. Proc Natl Acad Sci U S A 115: E7795–E7804 21. Das B, Dobrowolski C, Shahir AM, Feng Z, Yu X, Sha J, Bissada NF, Weinberg A, Karn J, Ye F (2015) Short chain fatty acids potently induce latent HIV-1 in T-cells by activating P-TEFb and multiple histone modifications. Virology 474:65–81 22. Bosque A, Famiglietti M, Weyrich AS, Goulston C, Planelles V (2011) Homeostatic proliferation fails to efficiently reactivate HIV-1 latently infected central memory CD4+ T cells. PLoS Pathog 7:e1002288 23. Ivanov II, RdL F, Manel N, Yoshinaga K, Rifkin DB, Sartor RB, Finlay BB, Littman DR (2008) Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 4:337–349 24. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman

DR (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121–1133 25. Sun H, Kim D, Li X, Kiselinova M, Ouyang Z, Vandekerckhove L, Shang H, Rosenberg ES, Yu XG, Lichterfeld M (2015) Th1/17 polarization of CD4 T cells supports HIV-1 persistence during antiretroviral therapy. J Virol 89: 11284–11293 26. Friedman J, Cho WK, Chu CK, Keedy KS, Archin NM, Margolis DM, Karn J (2011) Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2 (EZH2). J Virol 85:9078–9089 27. Mbonye UR, Gokulrangan G, Datt M, Dobrowolski C, Cooper M, Chance MR, Karn J (2013) Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4(+) T lymphocytes. PLoS Pathog 9:e1003338

Chapter 6 TGF-β Signaling Supports HIV Latency in a Memory CD4+ T Cell Based In Vitro Model Sydney Bergstresser and Deanna A. Kulpa Abstract During antiretroviral therapy (ART), HIV-1 persists as a latent reservoir in CD4+ T cell subsets in central (TCM), transitional (TTM) and effector memory (TEM) CD4+ T cells. Understanding the mechanisms that support HIV-1 latency in each of these subsets is essential to the identification of cure strategies to eliminate them. Due to the very low frequency of latently infected cells in vivo, model systems that can accurately reflect the heterogenous population of HIV-1 infected cells are a critical component in HIV cure discoveries. Here, we describe a novel primary cell-based model of HIV-1 latency that recapitulates the complex dynamics of the establishment and maintenance of the latent reservoir in different memory T cell subsets. The latency and reversion assay (LARA) culture conditions uniquely retain phenotypically and transcriptionally distinct memory CD4+ T cell subsets that allow in a single assay to assess LRA activity in each memory subset and differential examination of the dynamics of HIV latency reversal. Key words HIV-1, Latency, Memory CD4+ T cells

1

Introduction Despite continuous pharmaceutical treatment with ART, HIV-1 persists in infected individuals as latent provirus in a variety of immune cell types that express CD4 and CXCR4/CCR-5 coreceptors, such as CD4+ T lymphocytes, macrophages, dendrocytes, and microglial cells [1–6]. Together these latently infected cells form a cellular reservoir, which is capable of reestablishing viremia upon the cessation of ART [7–9]. The majority of proviral HIV DNA has been found in CD4+ T cells with a memory phenotype, and the long life span and quiescent phenotype of these cells represents a significant barrier to their elimination [7–13]. Studies to quantify the frequency of HIV-1-infected CD4+ T cells in vivo have suggested that there is approximately 1 replication-competent proviral genome per 106 CD4+ cells, which is estimated to equal 106 latently infected cells capable of generating replication competent virus in an individual on ART [14]. Given the long half-life of

Guido Poli et al. (eds.), HIV Reservoirs: Methods and Protocols, Methods in Molecular Biology, vol. 2407, https://doi.org/10.1007/978-1-0716-1871-4_6, © Springer Science+Business Media, LLC, part of Springer Nature 2022

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memory CD4+ T cells, it would require approximately 88 years of continuous suppressive therapy for the latently infected population to completely decay and eliminate the HIV-1 reservoir [15]. To overcome this barrier, efforts to develop cure strategies are targeted toward the exposure and elimination of cells harboring silent intact provirus. Latently infected cells escape immune system detection due to the suppression of HIV-1 transcription and viral antigen presentation that would normally signal for cytotoxicmediated removal [16]. Strategies such as “Shock and Kill” or “Kick and Kill” have been proposed to induce HIV-1 expression, which would reveal the infected cell to the immune system for elimination [17–20]. However, many challenges remain in studying HIV latency and viral reactivation, including the development of models that accurately predict the in vivo responses to HIV-1 cure strategies or time to viral rebound. The extremely low frequency of latently infected memory CD4+ T cells that harbor replication competent provirus makes it difficult to characterize these cells ex vivo [21]. Animal models offer an alternative to study mechanisms of viral latency in vivo. Infection of the nonhuman primate (NHP) rhesus macaque (Macaca mulatta) with SIV is widely used for studying HIV-1 infection and latency. However, NHP studies are costly, lengthy, and labor intensive. Advances in the development of HIV-1 latently infected cell lines have supported the identification of transcription factors and epigenetic modifications that are critical for modulation of virus expression, but have shown some limitations in the ability to cycle between active and quiescent states in response to biological stimuli [22– 25]. This phenotypic plasticity is important in the context of viral reactivation in vivo, and our ability to study latency mechanisms and latency reversal is critical for the development of a successful shock and kill strategy. Memory CD4+ T cells such as TCM, TTM, and TEM are endowed with very distinct activation, functional and survival properties, and expression of cell surface markers directs their homing to multiple tissues, such as the gut and lymph nodes [8, 11, 26– 29]. Significantly, all memory CD4+ T cell subsets have been shown to harbor persistent and replication competent HIV [7, 8, 11, 30, 31]. Given the heterogeneous nature of CD4+ T cell populations, strategic approaches to target the latent HIV-1 reservoir within these subsets will need to address the inherent molecular differences in TCM, TTM and TEM cells. The latency and reversion assay (LARA) is a primary cell based in vitro model that allows the monitoring of the establishment of HIV-1 latency and its reversion in different memory CD4+ T cell subsets [32]. Memory CD4+ T cells are enriched from HIV-naı¨ve PBMC and allowed to rest for 3 days prior to infection. Empirically, we have observed a resting period of 1–3 days increased the infectivity of the nonactivated memory CD4+ T cells used in LARA.

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71

After rest in culture, the memory CD4+ T cells are infected in vitro with replication competent clinical isolate HIV-1 89.6. In the development of LARA, multiple HIV-1 infectious clones were evaluated, including NL4-3. Other HIV-1 clones were able to infect the nonactivated memory CD4+ T cells in vitro, but the ability to establish a latent infection—and reverse latency—without significant cytopathic effects was highly variable. Spinoculation in the presence of hexadimethrine bromide (polybrene) is used to infect the memory CD4+ T cells. Immediately after infection, memory CD4+ T cells are put into culture with the protease inhibitor saquinavir. Saquinavir restricts HIV infection with the replication competent virus to a single round, which limits the accumulation of inactivating mutations or deletions that can influence the frequency of HIV latency reversal. The infected memory CD4+ T cells are again rested for several days to allow proviral DNA integration to come to completion. The HIV latency culture conditions in LARA were designed to mimic the homeostatic T cell environment in lymph nodes that allows the long-term maintenance of memory CD4+ T cells. LARA latency culture conditions include the addition of TGF-β, a cytokine with well-characterized potent inhibitory effects on T cell proliferation and differentiation [33–37]. IL-7 is also included, a γ-chain receptor cytokine that is involved in the maintenance of T cell memory and which has been shown to promote HIV persistence in vitro and in vivo [38, 39]. In vivo, TGF-β is produced by regulatory T cells (Tregs), a subset of cells known to be present in lymph nodes [40]. The antagonistic effect of TGF-β on IL-7 supports the maintenance of memory CD4+ T cells subsets without triggering increased viral replication [41]. An additional requirement of LARA is conditioned medium from the glioblastoma cell line H-80 [42, 43], which contains cytokines that promote quiescence (TGF-β 1, 2, and 3 and IL-9) and survival (IL-21; [44]). Importantly, LARA culture conditions also include addition of the antiretroviral compounds, besides saquinavir, two additional antiretroviral compounds are added to represent the multistep inhibition of viral propagation employed in vivo, the integrase inhibitor raltegravir, and the nonnucleoside reverse transcriptase inhibitor efavirenz to suppress viral spread and prevent preintegration latency. Memory CD4+ T cells can be maintained in LARA latency culture conditions for several weeks while monitoring for HIV-1 expression through flow cytometry [32]. It is of additional benefit to assess the frequency of cells carrying integrated proviral DNA by qPCR to confirm the maintenance of the latently infected cell population as HIV Gag expression declines [45]. Phenotypic assessment has shown that LARA conditions support the maintenance in vitro of all memory subsets as well as functional CD4+ T cell subsets (Th1, Th2, Th17) [32]. Significantly, transcriptional profiling showed that culture conditions used

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in LARA supported the maintenance of pathways that were specific to each memory T cell subset prior to initiation of cultures. Further analysis showed that perturbed pathways in this assay were mostly those downstream of TGF-β. These data support that LARA uniquely retains phenotypically and transcriptionally distinct memory CD4+ T cell subsets; a critical feature that allows the ability to assess LRA activity in each memory subset in a single assay [32]. After a minimum of one week in latency culture conditions, a population of highly infected memory CD4+ T cells are produced. Cells generated in LARA have been shown to respond to TCR engagement to a high level, with a majority of the latently infected cell population capable of reversing latency as detected by the upregulation of HIV Gag expression, both in the memory CD4+ population as a whole, as well as in TCM, TTM, and TEM. As part of validation of the LARA model, we studied the response of memory CD4+ T cells to several classes of LRAs including the protein kinase C agonist bryostatin, the HDAC inhibitors (HDACi) panobinostat, romidepsin, and SAHA, the acetaldehyde dehydrogenase inhibitor disulfiram, and the γ-c receptor cytokine IL-15. We compared the responsiveness to LRAs of latently infected cells generated in LARA to ex vivo samples from virally suppressed HIV-infected individuals and used TILDA, a quantitative PCR assay that measures the frequency of CD4+ T cells producing multispliced HIV RNA [46]. We found bryostatin had the highest latency reversal efficiency from both the latently infected cells generated in LARA (28%) and the HIV-infected ex vivo cells (19%) [32]. All other LRAs had low efficiency in reactivating HIV-1 from both LARA and ex vivo samples from HIV-1-infected cARTtreated individuals, including HDACi (90% CD133+ and/or CD34+. 133 S2 cells are typically >95% CD133+CD34+ and contain