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HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal.

ABSTRACT: OBJECTIVE:To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro. METHODS:Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified. RESULTS:Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8?±?1 and 2.9?±?0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1?±?0.6 and 1.8?±?0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts. CONCLUSION:HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.

SUBMITTER: Moso MA

PROVIDER: S-EPMC6319264 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal.

Moso Michael A MA Anderson Jenny L JL Adikari Samantha S Gray Lachlan R LR Khoury Georges G Chang Judy J JJ Jacobson Jonathan C JC Ellett Anne M AM Cheng Wan-Jung WJ Saleh Suha S Zaunders John J JJ Purcell Damian F J DFJ Cameron Paul U PU Churchill Melissa J MJ Lewin Sharon R SR Lu Hao K HK

AIDS (London, England) 20190201 2

<h4>Objective</h4>To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro.<h4>Methods</h4>Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the ...[more]

PMID: 30562171

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Project description:During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal.IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

Dataset Information

HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal.

Publications

HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal.

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