Cargando…

Entry of Polarized Effector Cells into Quiescence Forces HIV Latency

The latent HIV reservoir is generated following HIV infection of activated effector CD4 T cells, which then transition to a memory phenotype. Here, we describe an ex vivo method, called QUECEL (quiescent effector cell latency), that mimics this process efficiently and allows production of large numb...

Descripción completa

Detalles Bibliográficos
Autores principales: Dobrowolski, Curtis, Valadkhan, Saba, Graham, Amy C., Shukla, Meenakshi, Ciuffi, Angela, Telenti, Amalio, Karn, Jonathan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437053/
https://www.ncbi.nlm.nih.gov/pubmed/30914509
http://dx.doi.org/10.1128/mBio.00337-19
_version_ 1783406881750908928
author Dobrowolski, Curtis
Valadkhan, Saba
Graham, Amy C.
Shukla, Meenakshi
Ciuffi, Angela
Telenti, Amalio
Karn, Jonathan
author_facet Dobrowolski, Curtis
Valadkhan, Saba
Graham, Amy C.
Shukla, Meenakshi
Ciuffi, Angela
Telenti, Amalio
Karn, Jonathan
author_sort Dobrowolski, Curtis
collection PubMed
description The latent HIV reservoir is generated following HIV infection of activated effector CD4 T cells, which then transition to a memory phenotype. Here, we describe an ex vivo method, called QUECEL (quiescent effector cell latency), that mimics this process efficiently and allows production of large numbers of latently infected CD4(+) T cells. Naïve CD4(+) T cells were polarized into the four major T cell subsets (Th1, Th2, Th17, and Treg) and subsequently infected with a single-round reporter virus which expressed GFP/CD8a. The infected cells were purified and coerced into quiescence using a defined cocktail of cytokines, including tumor growth factor beta, interleukin-10 (IL-10), and IL-8, producing a homogeneous population of latently infected cells. Flow cytometry and transcriptome sequencing (RNA-Seq) demonstrated that the cells maintained the correct polarization phenotypes and had withdrawn from the cell cycle. Key pathways and gene sets enriched during transition from quiescence to reactivation include E2F targets, G(2)M checkpoint, estrogen response late gene expression, and c-myc targets. Reactivation of HIV by latency-reversing agents (LRAs) closely mimics RNA induction profiles seen in cells from well-suppressed HIV patient samples using the envelope detection of in vitro transcription sequencing (EDITS) assay. 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 4 years. The ease, efficiency, and accuracy of the mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.
format Online
Article
Text
id pubmed-6437053
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher American Society for Microbiology
record_format MEDLINE/PubMed
spelling pubmed-64370532019-04-03 Entry of Polarized Effector Cells into Quiescence Forces HIV Latency Dobrowolski, Curtis Valadkhan, Saba Graham, Amy C. Shukla, Meenakshi Ciuffi, Angela Telenti, Amalio Karn, Jonathan mBio Research Article The latent HIV reservoir is generated following HIV infection of activated effector CD4 T cells, which then transition to a memory phenotype. Here, we describe an ex vivo method, called QUECEL (quiescent effector cell latency), that mimics this process efficiently and allows production of large numbers of latently infected CD4(+) T cells. Naïve CD4(+) T cells were polarized into the four major T cell subsets (Th1, Th2, Th17, and Treg) and subsequently infected with a single-round reporter virus which expressed GFP/CD8a. The infected cells were purified and coerced into quiescence using a defined cocktail of cytokines, including tumor growth factor beta, interleukin-10 (IL-10), and IL-8, producing a homogeneous population of latently infected cells. Flow cytometry and transcriptome sequencing (RNA-Seq) demonstrated that the cells maintained the correct polarization phenotypes and had withdrawn from the cell cycle. Key pathways and gene sets enriched during transition from quiescence to reactivation include E2F targets, G(2)M checkpoint, estrogen response late gene expression, and c-myc targets. Reactivation of HIV by latency-reversing agents (LRAs) closely mimics RNA induction profiles seen in cells from well-suppressed HIV patient samples using the envelope detection of in vitro transcription sequencing (EDITS) assay. 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 4 years. The ease, efficiency, and accuracy of the mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency. American Society for Microbiology 2019-03-26 /pmc/articles/PMC6437053/ /pubmed/30914509 http://dx.doi.org/10.1128/mBio.00337-19 Text en Copyright © 2019 Dobrowolski et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Dobrowolski, Curtis
Valadkhan, Saba
Graham, Amy C.
Shukla, Meenakshi
Ciuffi, Angela
Telenti, Amalio
Karn, Jonathan
Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
title Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
title_full Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
title_fullStr Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
title_full_unstemmed Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
title_short Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
title_sort entry of polarized effector cells into quiescence forces hiv latency
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437053/
https://www.ncbi.nlm.nih.gov/pubmed/30914509
http://dx.doi.org/10.1128/mBio.00337-19
work_keys_str_mv AT dobrowolskicurtis entryofpolarizedeffectorcellsintoquiescenceforceshivlatency
AT valadkhansaba entryofpolarizedeffectorcellsintoquiescenceforceshivlatency
AT grahamamyc entryofpolarizedeffectorcellsintoquiescenceforceshivlatency
AT shuklameenakshi entryofpolarizedeffectorcellsintoquiescenceforceshivlatency
AT ciuffiangela entryofpolarizedeffectorcellsintoquiescenceforceshivlatency
AT telentiamalio entryofpolarizedeffectorcellsintoquiescenceforceshivlatency
AT karnjonathan entryofpolarizedeffectorcellsintoquiescenceforceshivlatency