Abstract
Antiretroviral therapy (ART) has transformed the deadly human immunodeficiency virus type I (HIV-1) epidemic into a manageable chronic condition. Current ART is not curative and treatment interruption leads to viral rebound in people living with HIV-1 (PLWH). The main cause of viral rebound is the persistence of HIV reservoirs in long-lived memory CD4+ T cells. Accurate techniques to identify and quantify viral reservoirs are required to monitor therapeutic approaches designed to cure HIV infection. Th17-polarized CD4+ T cells are located at mucosal sites of HIV entry and are preferentially targeted for infection and viral reservoir persistence. They constitute an important reservoir in both blood and colon. In this chapter we describe a step-by-step flow cytometry-based protocol to isolate a fraction of Th17-enriched cells from PBMC based on their expression of the Th17 surface marker CCR6. The isolation of memory CCR6+CD4+ T cells allows subsequent PCR/RT-PCR-based HIV DNA/RNA quantifications, as well as their culture for quantitative viral outgrowth assays (QVOA). This method can be adapted for the isolation of CCR6+CD4+ T cells from peripheral tissues, such as the colon.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 cells. Annu Rev Immunol 27:485–517
Sundrud MS, Trivigno C (2013) Identity crisis of Th17 cells: many forms, many functions, many questions. Semin Immunol 25(4):263–272
Dong C (2008) TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat Rev Immunol 8(5):337–348
Stockinger B, Omenetti S (2017) The dichotomous nature of T helper 17 cells. Nat Rev Immunol 17(9):535–544
Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ et al (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126(6):1121–1133
Huang W, Littman DR (2015) Regulation of RORgammat in inflammatory lymphoid cell differentiation. Cold Spring Harb Symp Quant Biol 80:257–263
Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B et al (2007) Phenotypic and functional features of human Th17 cells. J Exp Med 204(8):1849–1861
Wang C, Kang SG, Lee J, Sun Z, Kim CH (2009) The roles of CCR6 in migration of Th17 cells and regulation of effector T-cell balance in the gut. Mucosal Immunol 2(2):173–183
Wan Q, Kozhaya L, ElHed A, Ramesh R, Carlson TJ, Djuretic IM et al (2011) Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine production by CCR6+ human memory T cells. J Exp Med 208(9):1875–1887
Wacleche VS, Landay A, Routy JP, Ancuta P (2017) The Th17 lineage: from barrier surfaces homeostasis to autoimmunity, cancer, and HIV-1 pathogenesis. Viruses 9(10):303
Wacleche VS, Goulet JP, Gosselin A, Monteiro P, Soudeyns H, Fromentin R et al (2016) New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy. Retrovirology 13(1):59
Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gattorno M, Lanzavecchia A et al (2007) Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 8(6):639–646
Liang SC, Long AJ, Bennett F, Whitters MJ, Karim R, Collins M et al (2007) An IL-17F/a heterodimer protein is produced by mouse Th17 cells and induces airway neutrophil recruitment. J Immunol 179(11):7791–7799
Pelletier M, Maggi L, Micheletti A, Lazzeri E, Tamassia N, Costantini C et al (2010) Evidence for a cross-talk between human neutrophils and Th17 cells. Blood 115(2):335–343
Maxwell JR, Zhang Y, Brown WA, Smith CL, Byrne FR, Fiorino M et al (2015) Differential roles for Interleukin-23 and Interleukin-17 in intestinal Immunoregulation. Immunity 43(4):739–750
McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T et al (2007) TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol 8(12):1390–1397
Kebir H, Ifergan I, Alvarez JI, Bernard M, Poirier J, Arbour N et al (2009) Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis. Ann Neurol 66(3):390–402
Planas D, Routy JP, Ancuta P (2019) New Th17-specific therapeutic strategies for HIV remission. Curr Opin HIV AIDS 14(2):85–92
Planas D, Zhang Y, Monteiro P, Goulet JP, Gosselin A, Grandvaux N et al (2017) HIV-1 selectively targets gut-homing CCR6+CD4+ T cells via mTOR-dependent mechanisms. JCI Insight 2(15):e93230
Brenchley JM, Paiardini M, Knox KS, Asher AI, Cervasi B, Asher TE et al (2008) Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Blood 112(7):2826–2835
Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S et al (2006) Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 12(12):1365–1371
Estes JD, Harris LD, Klatt NR, Tabb B, Pittaluga S, Paiardini M et al (2010) Damaged intestinal epithelial integrity linked to microbial translocation in pathogenic simian immunodeficiency virus infections. PLoS Pathog 6(8):e1001052
Gosselin A, Wiche Salinas TR, Planas D, Wacleche VS, Zhang Y, Fromentin R et al (2017) HIV persists in CCR6+CD4+ T cells from colon and blood during antiretroviral therapy. AIDS 31(1):35–48
Anderson JL, Khoury G, Fromentin R, Solomon A, Chomont N, Sinclair E et al (2020) Human immunodeficiency virus (HIV)-infected CCR6+ rectal CD4+ T cells and HIV persistence on antiretroviral therapy. J Infect Dis 221(5):744–755
Zhang Y, Planas D, Raymond Marchand L, Massanella M, Chen H, Wacleche V et al (2020) Improving HIV outgrowth by optimizing cell-culture conditions and supplementing with all-trans retinoic acid. Front Microbiol 11:902
Falcinelli SD, Ceriani C, Margolis DM, Archin NM (2019) New Frontiers in measuring and characterizing the HIV reservoir. Front Microbiol 10:2878
Planas D, Fert A, Zhang Y, Goulet JP, Richard J, Finzi A et al (2020) Pharmacological inhibition of PPARγ boosts HIV reactivation and Th17 effector functions, while preventing progeny virion release and de novo infection. Pathog Immun 5(1):177–239
Cleret-Buhot A, Zhang Y, Planas D, Goulet JP, Monteiro P, Gosselin A et al (2015) Identification of novel HIV-1 dependency factors in primary CCR4(+)CCR6(+)Th17 cells via a genome-wide transcriptional approach. Retrovirology 12:102
Monteiro P, Gosselin A, Wacleche VS, El-Far M, Said EA, Kared H et al (2011) Memory CCR6+CD4+ T cells are preferential targets for productive HIV type 1 infection regardless of their expression of integrin beta7. J Immunol 186(8):4618–4630
Roederer M (2001) Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45(3):194–205
Acknowledgments
The authors acknowledge the contribution of Dr. Dominique Gauchat (Flow Cytometry Core Facility, CHUM-Research Centre, Montreal, QC, Canada) for expert technical support with flow cytometry analysis and sorting, Mario Legault for help with ethical approvals and informed consents, Dr. Jean-Pierre Routy and Mrs. Josée Girouard, Angie Massicotte, and Maria Fraraccio (McGill University Health Centre—Glen Site, Montreal, QC, Canada) for their contribution to blood collection from HIV-infected study participants. The authors thank Mrs. Laurence Raymond Marchand for the critical revision of this manuscript, as well as Mrs. Annie Gosselin and Drs. Patricia Monteiro, Aurélie Cleret-Buhot, and Vanessa Sue Wacleche for setting up the initial CCR6+ T-cell isolation protocol. Finally, the authors thank biological sample donors for their major contribution to this work.
Funding: This study was supported by grants to PA from the Canadian Institutes of Health Research (CIHR; #MOP-114957; #PJT-153052; IBC-154053); the Fonds de Recherche du Québec-Santé (FRQ-S)/AIDS and Infectious Diseases Network, Québec, Canada; The Canadian HIV Cure Enterprise Team Grant (CanCURE 1.0) funded by the CIHR in partnership with CANFAR and IAS (CanCURE 1.0; # HIG-133050), and The Canadian HIV Cure Enterprise Team Grant (CanCURE 2.0) funded by the CIHR (#HB2–164064).
Conflict of Interest: The authors declare no conflicts of interest.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Cattin, A., Fert, A., Planas, D., Ancuta, P. (2022). Flow Cytometry Sorting of Memory CCR6+CD4+ T-Cells for HIV Reservoir Quantification. In: Poli, G., Vicenzi, E., Romerio, F. (eds) HIV Reservoirs. Methods in Molecular Biology, vol 2407. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1871-4_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1871-4_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1870-7
Online ISBN: 978-1-0716-1871-4
eBook Packages: Springer Protocols