Related Experiment Video
Updated: Aug 10, 2025
Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis
Published on: September 7, 2022
Automated microarray for single-cell sorting and collection of lymphocytes following HIV reactivation
Insights
Understanding HIV reactivation is key to a cure. New methods reveal single-cell differences in how latent HIV responds to latency reversing agents (LRAs), guiding future HIV cure strategies.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- A potential HIV cure involves reactivating latent viruses with latency reversing agents (LRAs) and eliminating infected cells.
- Incomplete and heterogeneous HIV reactivation from latency hinders complete viral eradication.
- Understanding single-cell HIV reactivation mechanisms is crucial for developing effective cure strategies.
Approach:
- Microraft arrays were utilized to image T-lymphocytes and track HIV long terminal repeat (LTR) promoter activity over time.
- Cells were exposed to various LRAs (prostratin, iBET151, SAHA) to assess their impact on viral reactivation.
- Single-cell RNA-sequencing was performed on sorted fast and slow-reactivating T-lymphocytes to analyze molecular differences.
Key Points:
- LRAs induced heterogeneous HIV reactivation, with varying kinetics and fluorescence levels observed at the single-cell level.
- Prostratin demonstrated distinct subpopulations of T-lymphocytes exhibiting slow and fast reactivation kinetics.
- Single-cell RNA-sequencing identified differential gene expression related to inflammation, immune activation, and transcription factors in reactivator subpopulations.
Conclusions:
- This study provides critical insights into the heterogeneity of HIV reactivation dynamics at the single-cell level.
- The findings advance the conceptual understanding of HIV latency reversal, essential for designing more effective therapeutic interventions.
- The developed methodology enables detailed analysis of single-cell responses to LRAs, paving the way for optimized HIV cure strategies.
Abstract:
A promising strategy to cure HIV infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of various LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5 %, 11.2 %, and 12.1 % percentage of cells respectively, reactivated similar to that observed in other experimental systems. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found. These results advance our conceptual understanding of HIV reactivation dynamics at the single-cell level toward a cure for HIV.
More Related Videos
09:07Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations
Published on: August 22, 2019
07:21Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment
Published on: June 23, 2019